Methods and compositions for diagnosing, prognosing, and treating endometriosis

ABSTRACT

This document provides methods and materials related to genetic variations associated with endometriosis. For example, this document provides methods for using such genetic variations to assess risk of, or susceptibility of developing or diagnosing endometriosis.

The instant application includes a file identified as follows:“3886001US1sequencelisting.txt”, which is 22,601,728 bytes in size. Thisfile contains tabulated sequence information in non-delineated format.The aforementioned file was created on Nov. 3, 2014 and is herebyincorporated by reference in its entirety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/197,593, filed Nov. 21, 2018, which is a divisional of U.S. patentapplication Ser. No. 14/538,404 filed Nov. 11, 2014, (now U.S. Pat. No.10,174,376), issued on Jan. 8, 2019, which claims the benefit of U.S.Provisional Application No. 61/903,286, filed on Nov. 12, 2013, thedisclosure of which is incorporated by reference herein.

BACKGROUND

Endometriosis is an estrogen-dependent gynecologic disorder, defined asthe presence of endometrial-like tissue outside the uterine cavity,which affects 6% to 10% women of reproductive age from all ethnic andsocial groups. The degree of endometriosis is staged according to theclassification system of the American Society of Reproductive Medicine(Fertil. Steril., 67:81721 (1997)) into minimal, mild, moderate, andsevere disease. At present, the gold standard for diagnosis ofendometriosis is laparoscopic inspection with histologic confirmationafter retrieval of lesions.

As endometriosis can be progressive in up to 50% of women, early,noninvasive diagnosis has the potential to offer early treatment andprevent progression. A noninvasive test for endometriosis would be alsouseful for women with pelvic pain and/or subfertility with normalultrasound results. The goal of a non-invasive test is that no womenwith endometriosis or other significant pelvic pathology are missed whomight benefit from medical therapy or surgery. There are reports ofbiomarkers that may be useful for early or noninvasive detection. Forexample, Fossbinder et al (Fertil. Steril. 99:1135-1145 (2013)) pointout that cancer antigen 125 (CA-125) is the most used peripheralbiomarker of endometriosis, and that out of 28 biomarkers, multivariateanalysis of plasma samples, showed that annexin V, vascular endothelialgrowth factor (VEGF), CA-125, and soluble intercellular adhesionmolecule-1 (sICAM-1)/or glycodelinin enabled the diagnosis ofendometriosis in women who had disease undetected by ultrasound.Surprisingly, inflammatory molecules did not emerge as biomarkers in apanel with the best diagnostic performers.

May et al. (Hum. Reprod. Update. 17:637-53 (2011)) conducted asystematic review of published results that assessed over 200 potentialbiomarkers for endometriosis, including hormones and their receptors,cytokines, and factors identified using proteomics, and analyzedhistological results from endometrial tissue. Some of those putativebiomarkers related to nerve fiber growth or cell cycle control, whileothers were cytokines including IL-1β, IL-IR type II, IL-6, IL-8, IL-13,IL-15, tumor necrosis factor (TNF)-α, (MCP-1); macrophage-stimulatingprotein (RANTES); steroids and hormones, e.g., aromatase andhydroxysteroid dehydrogenase enzymes; growth factors; e.g., TGFβ family,insulin-like growth (IGF) factors, hepatocyte growth factor (IMF) andits receptor, annexin-1; cell adhesion and extracellular matrixmolecules, e.g., the r33 integrin subunit α₃β₁ integrin, α_(v)β₅ andα_(v)β₅ integrins, E-cadherin; extracellular matrix molecules (ECM),e.g., ICAM-1, focal adhesion kinase (FAK); tissue remodeling molecules,e.g., matrix metalloproteinase (MMP) family, tissue inhibitors ofmetalloproteinases (TIMPs), urokinase; angiogenesis, e.g., vascularendothelial growth factor (VEGF), angiopoietin-1 and -2 molecules;associated with apoptosis and cell cycle control, e.g., calpain 5,MCL-1, Bak, Ki67, telomerase activity, proliferating cell nuclearantigen, Pak-1, phosphorylated ERK 112, c-myc, survivin; reactive oxygenand nitrogen species, e.g., endothelial xanthine oxidase, WT-1, CCL16,CCL21, HOXA10 and COX-2.

SUMMARY OF THE INVENTION

The invention provides a method of screening one or more female subjectsfor those having endometriosis (EN), those with altered susceptibilityto developing EN or those at risk of developing EN. The method comprisesassaying at least one genetic sample of one or more subjects, nucleicacid sequence information from the one or more subjects, or providingthat information, for at least one genetic variation in one or more lociassociated with EN, e.g., gene variations associated with one or moregenes in FIG. 3 . The presence in the genetic sample of the at least onegenetic variation is used to determine whether the one or more subjectshave EN, have an altered susceptibility to EN or are at risk of EN. Insome embodiments, determining whether the one or more subjects have EN,are at risk of EN or have an altered susceptibility to EN includes agynecological examination and/or medical history analysis of the one ormore subjects, e.g., in addition to the nucleic acid sequenceinformation. In some embodiments, at least one genetic sample iscollected from blood, e.g., peripheral blood mononuclear cells (PBMC) orperipheral blood lymphocytes (PBL), saliva, urine, serum, tears, skin,tissue, or hair from at least one subject. In some embodiments, assayingthe at least one genetic sample of one or more subjects includespurifying the at least one genetic sample. In some embodiments, assayingthe at least one genetic sample of the one or more subjects includesamplifying at least one nucleotide or a specific region of one or morechromosomes in the at least one genetic sample. In some embodiments,assaying the at least one genetic sample of the one or more subjectsincludes assaying an unamplified sample for at least one nucleotide or aspecific region of one or more chromosomes in the at least one geneticsample. In some embodiments, assaying the at least one genetic samplefor at least one genetic variation includes a microarray analysis of theat least one sample. In some embodiments, the microarray analysiscomprises a comparative genomic hybridization (CGH) array analysis.

In one embodiment, the invention provides a method of diagnosing asusceptibility to endometriosis in a female subject. The method includesproviding nucleic acid sequence information from the female subject onthe presence or absence of at least genetic variation in one or moregenes or regions in FIG. 1 or FIG. 2 . A susceptibility to endometriosisin the subject is diagnosed if the subject has at least one geneticvariation in the one or more genes or regions in FIG. 1 , wherein the atleast genetic variation occurs in the gene or region more frequently ina population of female subjects that have endometriosis than in apopulation of female subjects that does not have endometriosis. In oneembodiment, the nucleic acid sequence information is for one or moregenes or regions that include but are not limited to one or more ofTGFBR3, FUT9, PDE1C, IMPK, GIGYF2, HMGB3, ZFP14, ACCS, or DPP6. In oneembodiment, the nucleic acid sequence information is for one or moregenes or regions that include but are not limited to one or more ofMYO1B, MIR3675, NBPF1, or GPR111. In one embodiment, the nucleic acidsequence information is for one or more genes or regions that includebut are not limited to one or more of TGFB1I1, PTK2, PGRMC2, LEPROT,LEPR, MUC4, MAGEA11, BOK, BOK-AS1, TSHR, MSN, MYADML, CYP17A1, RXFP1,CRHR2, PLA2G4C, NCOA1, BNC2, or MKRN1. In one embodiment, the nucleicacid sequence information is for one or more genes or regions thatinclude but are not limited to one or more of ARMC5, C16orf58, SLC5A2,ZNF843, LARP1B, DNAJC6, AS3MT, C10orf32, AS3MT-C10orf32, CNNM2, orRAB19.

In one embodiment, the invention provides a method of diagnosing asusceptibility to endometriosis in a female subject. The method includesdetecting in a sample of the subject nucleic acid sequence informationon the presence or absence of at least one genetic variation in one ormore regions in FIG. 2 . A susceptibility to endometriosis is diagnosedin the subject if the subject has at least one genetic variation in theone or more genes or regions in FIG. 1 or FIG. 2 , wherein the at leastgenetic variation occurs in the gene or region more frequently in apopulation of female subjects that have endometriosis than in apopulation of female subjects that does not have endometriosis. In oneembodiment, the nucleic acid sequence information is for one or moregenes or regions that include but are not limited to one or more ofTGFBR3, FUT9, PDE1C, IMPK, GIGYF2, HMGB3, ZFP14, ACCS, or DPP6. In oneembodiment, the nucleic acid sequence information is for one or moregenes or regions that include but are not limited to one or more ofMYO1B, MIR3675, NBPF1, or GPR111. In one embodiment, the nucleic acidsequence information is for one or more genes or regions that includebut are not limited to one or more of TGFB1I1, PTK2, PGRMC2, LEPROT,LEPR, MUC4, MAGEA11, BOK, BOK-AS1, TSHR, MSN, MYADML, CYP17A1, RXFP1,CRHR2, PLA2G4C, NCOA1, BNC2, or MKRN1. In one embodiment, the nucleicacid sequence information is for one or more genes or regions thatinclude but are not limited to one or more of ARMC5, C16orf58, SLC5A2,ZNF843, LARP1B, DNAJC6, AS3MT, C10orf32, AS3MT-C10orf32, CNNM2, orRAB19.

In one embodiment, the invention provides a method that includesproviding nucleic acid sequence information from a female subject on thepresence or absence of at least genetic variation in one or more genesor regions in FIG. 2 . A susceptibility to endometriosis in the subjectis diagnosed if the subject has at least one genetic variation in theone or more genes or regions in FIG. 2 , wherein the at least geneticvariation occurs in the gene or region more frequently in a populationof female subjects that have endometriosis than in a population offemale subjects that does not have endometriosis. In one embodiment, thenucleic acid sequence information is for one or more genes or regionsthat include but are not limited to one or more of TGFBR3, FUT9, PDE1C,IMPK, GIGYF2, HMGB3, ZFP14, ACCS, or DPP6. In one embodiment, thenucleic acid sequence information is for one or more genes or regionsthat include but are not limited to one or more of MYO1B, MIR3675,NBPF1, or GPR111. In one embodiment, the nucleic acid sequenceinformation is for one or more genes or regions that include but are notlimited to one or more of TGFB1I1, PTK2, PGRMC2, LEPROT, LEPR, MUC4,MAGEA11, BOK, BOK-AS1, TSHR, MSN, MYADML, CYP17A1, RXFP1, CRHR2,PLA2G4C, NCOA1, BNC2, or MKRN1. In one embodiment, the nucleic acidsequence information is for one or more genes or regions that includebut are not limited to one or more of ARMC5, C16orf58, SLC5A2, ZNF843,LARP1B, DNAJC6, AS3MT, C10orf32, AS3MT-C10orf32, CNNM2, or RAB19.

In some embodiments, the method further comprises designing or preparingan array, e.g., a CGH array, to measure or detect one or more geneticvariations in the regions shown in FIG. 1 or FIG. 2 . In someembodiments, the method further comprises providing such a CGH array forthe measuring or detecting of one or more genetic variations. In someembodiments, assaying at least one genetic sample comprises obtainingnucleic acid sequence information. In some embodiments, obtaining thenucleic acid sequence information is accomplished by one or more methodsincluding but not limited to PCR, sequencing, Northern blots, multiplexligation-dependent probe amplification (MLPA), molecular beacon, arrayComparative Genomic Hybridization, Invader assay, ligase chain reaction(LCR), fluorescence in situ hybridization, or any combination thereof.In some embodiments, sequencing comprises one or more high-throughputsequencing methods.

In some embodiments, determining whether one or more test subjects haveEN, are at risk of EN or have an altered susceptibility to EN includescomparing the nucleic acid sequence information of the one or more testsubjects, the at least one genetic variation identified in the one ormore test subjects, or a combination thereof, to those of one or morecontrol subjects, e.g., subjects that do not have EN, are not at risk ofEN or do not have an enhanced susceptibility to EN. In some embodiments,the one more control subjects include one or more subjects not suspectedof having EN and the one or more test subjects include one or moresubjects suspected of having EN. In some embodiments, the one or moretest subjects include one or more subjects with EN, and the one or morecontrol subjects include one or more subjects without EN. In someembodiments, the one or more test subjects include one or more subjectswho are symptomatic for EN, and the one or more control subjects includeone or more subjects who are asymptomatic for EN. In some embodiments,the one or more test subjects include one or more subjects that do notpresent with pain as a major symptom. In some embodiments, the one ormore test subjects have infertility issues (e.g., up to about 40% ofwomen with EN have infertility issues but have no or little pain relatedto their EN lesions). In some embodiments, the one or more controlsubjects include one or more subjects that have increased or decreasedsusceptibility to EN. In some embodiments, the one or more controlsubjects include one or more subjects associated or unassociated with atreatment, therapeutic regimen, or any combination thereof.

In some embodiments, determining whether the one or more test subjectshave EN, are at risk of EN or have an altered susceptibility to ENincludes comparing a gynecological examination, a medical historyanalysis, or a combination thereof, of the one or more test subjects tothe nucleic acid sequence information of the one or more test subjects,at least one genetic variation identified in the one or more testsubjects, the nucleic acid sequence information of one or more controlsubjects, at least one genetic variation identified in the one or morecontrol subjects, or a combination thereof.

In some embodiments, the at least one genetic variation comprises one ormore point mutations, single nucleotide polymorphisms (SNPs), singlenucleotide variants (SNVs), polymorphisms, translocations, insertions,deletions, amplifications, inversions, microsatellites, interstitialdeletions, copy number variations (CNVs), loss of heterozygosity, or anycombination thereof. For example, in some embodiments, the at least onegenetic variation includes one or more genetic variations, e.g., CNVs inthe genes listed in FIG. 1 , e.g., genes having any one of SEQ IDNOs:1-47 or one or more genetic variations in CNV subregions listed inFIG. 2 . In some embodiments, the genetic variation includes one or moregenetic variations, e.g., CNVs, that disrupt or modulate one or moregenes listed in FIG. 3 . In some embodiments, the at least one geneticvariation includes variations such as one or more CNVs that disrupt ormodulate the expression or function of one or more RNA transcripts inFIG. 4 , e.g., those having any one of SEQ ID NOs:48-149

In one aspect, the invention provides a method for screening for atherapeutic agent useful for preventing, inhibiting or treating EN. Themethod includes identifying an agent that modulates the function orexpression of one or more genes listed in FIG. 3 or expression productstherefrom, or one or more RNA transcripts mentioned in FIG. 4 orexpression products thereof. In some embodiments, the expressionproducts include one or more proteins expressed from a gene listed inFIG. 3 or encoded by one or more transcripts mentioned in FIG. 4 . Insome embodiments, modulating the function or activity of one or more RNAtranscripts or proteins results in an increase in expression. In someembodiments, modulating the function or activity of one or more RNAtranscripts or proteins results in a decrease in expression.

In one aspect, a method of preventing, inhibiting or treating EN in asubject is provided. The method includes administering one or moreagents effective to modulate the function of one or more genes listed inFIG. 3 , or expression products therefrom, or one or more RNAtranscripts mentioned in FIG. 4 , or expression products thereof,thereby preventing, inhibiting or treating the EN. In some embodiments,the expression products are one or more proteins expressed from a genelisted in FIG. 3 , or encoded by one or more RNA transcripts mentionedin FIG. 4 or genes in the same pathway (see, e.g., FIG. 9 ). In someembodiments, the one or more agents include but are not limited to aprotein, e.g., an antibody, a drug, a combination of drugs, a compound,a combination of compounds, radiation, a genetic sequence, a combinationof genetic sequences, heat or cryogenics, or a combination of two ormore of any combination thereof.

In one aspect, a method for screening for a therapeutic agent useful fortreating EN is provided. The method includes identifying an agent thatmodulates the function or expression of one or more genes listed in FIG.3 or expression products therefrom. In some embodiments, the expressionproducts include one or more RNA transcripts in FIG. 4 . In someembodiments, the expression products include one or more proteinsexpressed from a gene listed in FIG. 3 or encoded by one or more RNAtranscripts in FIG. 4 . In some embodiments, modulating the function oractivity of one or more RNA transcripts or proteins includes an increasein expression. In some embodiments, modulating the function or activityof one or more RNA transcripts or proteins includes a decrease inexpression. In some embodiments, screening the one or more subjects alsoincludes selecting one or more therapies based on the presence orabsence of the one or more genetic variations, e.g., the presence of agenetic variation in at least one gene listed in FIG. 3 .

In one aspect, a method of treating a subject for EN is provided. Themethod includes administering one or more agents effective to modulatethe function of one or more genes listed in FIG. 3 , or expressionproducts therefrom, thereby treating EN. In some embodiments, theexpression products include one or more RNA transcripts in FIG. 4 . Insome embodiments, the expression products include one or more proteinsexpressed from a gene in FIG. 3 , or encoded by one or more RNAtranscripts in FIG. 4 . In some embodiments, the agent may be anantibody, a compound, a combination of compounds, radiation, a geneticsequence, a combination of genetic sequences, heat, cryogenics, and acombination of two or more of any combination thereof.

As described in Examples 3 and 4, CNV analysis using methods describedin Example 1 revealed the presence of a 4-probe spanning heterozygousTGFBR3 deletion in 3 individuals with endometriosis. Primer pairsspecific for that region successfully generated a product of theexpected size in the deletion carriers but not in either normal DNA orDNA from an endometriosis patient without the deletion. Thus, TGFBR3genetic variations, e.g., the TGFBR3 CNV or others described herein, maybe used in an assay that would facilitate rapid and low cost screeningof endometriosis cohorts for the presence of the genetic variation,e.g., deletion, in order to obtain better estimates for the frequency ofthe variation in such cohorts and diagnose the cause of endometriosis inthose who carry the variation.

The method comprises assaying at least one genetic sample of one or moresubjects, nucleic acid sequence information from the one or moresubjects, or providing that information, for at least one geneticvariation impacting or encompassing TGFBR3. The presence in the geneticsample of the at least one genetic variation is used to determinewhether the one or more subjects have EN, have an altered susceptibilityto EN or are at risk of EN. In some embodiments, determining whether theone or more subjects have EN, are at risk of EN or have an alteredsusceptibility to EN includes a gynecological examination and/or medicalhistory analysis of the one or more subjects, e.g., in addition to thenucleic acid sequence information. In some embodiments, at least onegenetic sample is collected from blood, e.g., peripheral bloodmononuclear cells (PBMC) or peripheral blood lymphocytes (PBL), saliva,urine, serum, tears, skin, tissue, or hair from at least one subject. Insome embodiments, assaying the at least one genetic sample of one ormore subjects includes purifying the at least one genetic sample. Insome embodiments, assaying the at least one genetic sample of the one ormore subjects includes amplifying at least one nucleotide or a specificregion of one or more chromosomes in the at least one genetic sample. Insome embodiments, assaying the at least one genetic sample of the one ormore subjects includes assaying an unamplified sample for at least onenucleotide or a specific region of one or more chromosomes in the atleast one genetic sample. In some embodiments, assaying the at least onegenetic sample for at least one genetic variation includes a microarrayanalysis of the at least one sample. In some embodiments, the microarrayanalysis comprises a comparative genomic hybridization (CGH) arrayanalysis. In one embodiment, the method includes detecting a deletion inTGFBR3, e.g., using a multiplex ligation-dependent probe amplification(MLPA), molecular beacon, aCGH, Invader assay, ligase chain reaction(LCR), or fluorescence in situ hybridization.

In one aspect, the invention provides a kit for screening for EN in asubject. The kit includes at least one component for assaying a geneticsample from the subject for the presence of at least one geneticvariation in the genes listed in FIG. 1 or in FIG. 2 associated with EN.In one embodiment, a kit to screen for the TGFBR3 deletion, as describedin Example 4, contains PCR primers such as Example 4 OUTER_FWD andOUTER_REV primers or similar primer pairs (see below) that yield aspecific amplification product in genetic samples that contain theTGFBR3 deletion but do not yield an amplification product in geneticsamples without the TGFBR3 deletion. In another embodiment, a kit toscreen for the TGFBR3 deletion, as described in Example 4, contains anInvader oligonucleotide and primary probe that target the specificjunction fragment of DNA sequence resulting from the deletion andproduce a signal in genetic samples that contain the TGFBR3 deletion butdo not produce a signal in genetic samples without the TGFBR3 deletion.In some embodiments, the at least one genetic variation is associatedwith a disruption or aberration of one or more RNA transcripts mentionedin FIG. 4 . In some embodiments, the at least one genetic variation isassociated with a disruption or aberration of one or more proteinsexpressed from one or more genes listed in FIG. 3 , or encoded by one ormore RNA transcripts mentioned in FIG. 4 . One embodiment provides a kitfor screening for endometriosis in one or more female subjects, the kitcomprising reagents for assaying a genetic sample from the one or moresubjects for the presence or absence of at least one genetic variationin one or more genes or regions in FIG. 1 or 3 , or a combinationthereof.

In some embodiments, screening the one or more subjects furthercomprises selecting one or more therapies based on the presence orabsence of the one or more genetic variations. In some embodiments, thenucleic acid sequencing information is obtained for the whole genome orwhole exome from the one or more subjects. In some embodiments, thenucleic acid sequencing information has already been obtained for thewhole genome or whole exome from the one or more individuals and thenucleic acid information is obtained from in silico analysis. In otherembodiments, the nucleic acid sequencing information is obtained for aselected portion of the whole genome or whole exome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary regions with genetic variations that areassociated with EN. For each variation in each EN subject, the followingare provided: chromosome for the variation, start and stop locations fororiginal CNV; original CNV size; CNV type; subject case identifier (ID);gene symbol; and SEQ ID number corresponding to that region.

FIG. 2 shows exemplary CNV subregions with genetic variations that areassociated with EN. For each variation in each EN case, the followingare provided: chromosome for the variation; start and stop location ofCNV subregion; CNV subregion size; CNV type; subject case ID(s); genesymbol; whether the CNV subregion overlaps an exon; the number ofcontrol (NVE) subjects with a CNV in that subregion, the number of ENcases with a CNV in that subregion; Fisher 2-tailed Exact Test (FET);odds ratio (OR); and whether the CNV subregion is of interest due to FETor OR, or a biological association with EN.

FIG. 3 summarizes the characteristics of genes in the regions associatedwith EN.

FIG. 4 summarizes transcripts in the regions associated with EN andcorresponding SEQ ID Nos.

FIG. 5 schematic of deletion in chromosome 1 within TGFBR3 that is foundin 3 EN cases and 0 controls.

FIG. 6 schematic of insertion in chromosome 7 within PDE1C that is foundin 3 EN cases and 0 controls.

FIG. 7 shows amplification products from the use of a primer set todetect a deletion in TGFRB3. M=marker (Bioline Hyperladder I—200 bp, 400bp, 600 bp, 800 bp, 1000 bp (bold), 1.5 kb, 2 kb, 2.5 kb, 3 kb, 4 kb, 5kb, 6 kb, 8 kb, and 10 kb). N=No template control. A=D-001739 template(30 ng). Contains the deletion. B=D-002732 template (30 ng). Containsthe deletion. C=D-003697 template (30 ng). Contains the deletion.D=D-001832 template (30 ng). Does not contain the deletion. P=Biolinepooled placental genomic DNA (30 ng).

FIG. 8 is a BLAT analysis of Fwd sequence from 3 unrelated endometriosiscases with an approximately 8 kb deletion in an intron of TGFBR3,demonstrating the identical nature of the deletion in all 3 cases andthe presence of 4 probes (as observed in the CNV array data herein, seeFIG. 5 wherein the 4 probes define a minimal deletion size ofapproximately 6.8 kb) within the deletion.

FIG. 9 shows molecular interactions on the basis of biochemical and insilico evidence generated with the String 9.05 algorithm(http://string-db.org/). Four genes (PDE1C, PTK2, TGFBR3, TBFB1I1) thatwere identified on the basis of CNVs present in 1-3 EN patients and 0controls are indicated along with OR values. Genes with EN-relevantbiology are encircled.

FIG. 10 provides a list of genes that are known drug targets as reportedin Agarwal et al., Nat. Rev. Drug Discov. 12(8):575-6 (2013). Categorydefines the target as ‘proven’ if at least one drug selective for itsprotein product is approved and ‘novel’ if no drug has been approvedyet. Gene lists the gene symbol for the drug target, latest phase liststhe latest stage of drug development for the target, and competitionlists the number of pharmaceutical companies that have a drug indevelopment or launched for a given drug target.

FIG. 11 represents an example of a typical result in a heterozygousdeletion carrier and a non-carrier.

DETAILED DESCRIPTION

Genetic risk can be conferred by subtle differences in individualgenomes within a population. Genes can differ between individuals due togenomic variability, and the most frequent differences are due to singlenucleotide polymorphisms (SNPs). SNPs can be located, on average, every500-1000 base pairs in the human genome. Additional geneticpolymorphisms in a human genome can be caused by duplication, insertion,deletion, translocation and/or inversion, of short and/or long stretchesof DNA. Thus, in general, genetic variability among individuals occurson many scales, ranging from single nucleotide changes, to gross changesin chromosome structure and function. Many copy number variations (CNVs)of DNA segments, including deletions, insertions, duplications andcomplex multi-site variants, ranging in length from kilobases tomegabases in size, have been discovered (Redon et al., Nature,444:444-54 (2006) and Estivill & Armengol, PLoS Genetics, 3:1787(2007)). Known CNVs account for over 15% of the assembled human genome(Estivill & Armengol, supra). However, a majority of these variants areextremely rare and cover a small percentage of a human genome of anyparticular individual.

Described herein are methods of identifying variations in nucleic acidsand genes associated with EN and their use in diagnosis, prognosis andtheranosis. Also described herein are methods of screening fordetermining a subject's risk of or susceptibility to developing EN, ormethods of diagnosing EN, based on identification and detection, ordetection, of genetic nucleic acid variations. Also described herein aremethods and compositions for treating, inhibiting and/or preventing ENusing a therapeutic modality. The present disclosure further encompassesmethods of assessing an individual for probability of response to atherapeutic agent for EN, methods for predicting the effectiveness of atherapeutic agent for EN, nucleic acids, polypeptides and antibodiesuseful in methods or kits, and computer-implemented functions. Kits forscreening a sample from a subject to detect or determine a risk of orsusceptibility to EN are also encompassed by the disclosure.

Genetic Variations Associated with Endometriosis

Genomic sequences within populations exhibit variability betweenindividuals at many locations in the genome. For example, the humangenome exhibits sequence variations, which occur on average every 500base pairs. Such genetic variations in nucleic acid sequences arecommonly referred to as polymorphisms or polymorphic sites. In someembodiments, these genetic variations can be found to be associated withEN using the methods disclosed herein. In some embodiments, thesegenetic variations comprise point mutations, e.g., single nucleotidepolymorphisms (SNPs) or single nucleotide variants (SNVs),polymorphisms, translocations, insertions, deletions, amplifications,inversions, interstitial deletions, copy number variations (CNVs), lossof heterozygosity, or any combination thereof. In some embodiments,polymorphisms (e.g., polymorphic markers, genetic variations, or geneticvariants) can comprise any nucleotide position at which two or moresequences are possible in a subject population. In some embodiments,each version of a nucleotide sequence with respect to the polymorphismcan represent a specific allele of the polymorphism. In someembodiments, genomic DNA from a subject can contain two alleles for anygiven polymorphic marker, representative of each copy of the marker oneach chromosome. In some embodiments, an allele can be a nucleotidesequence of a given location on a chromosome. Polymorphisms can compriseany number of specific alleles. In some embodiments of the disclosure, apolymorphism can be characterized by the presence of two or more allelesin a population. In some embodiments, the polymorphism can becharacterized by the presence of three or more alleles. In someembodiments, the polymorphism can be characterized by four or morealleles, five or more alleles, six or more alleles, seven or morealleles, nine or more alleles, or ten or more alleles. In someembodiments an allele can be associated with one or more diseases ordisorders. In some embodiments, genetic variations and alleles can beused to associate an inherited phenotype, for example, susceptibilityEN, with a responsible genotype. In some embodiments, an allele, e.g., arisk allele, can be a variant allele that is statistically associatedwith EN, a risk of developing EN, or an increase susceptibility to EN.In some embodiments, genetic variations can be of any measurablefrequency in the population, for example, a frequency higher than 10%, afrequency between 5-10%, a frequency between 1-5%, or frequency below1%. As used herein, variant alleles can be alleles that differ from areference allele. As used herein, a variant can be a segment of DNA thatdiffers from the reference DNA, such as a genetic variation. In someembodiments, genetic variations can be used to track the inheritance ofa gene that has not yet been identified, but whose approximate locationis known.

As used herein, a haplotype can be information regarding the presence orabsence of one or more genetic markers in a given chromosomal region ina subject. In some embodiments, a haplotype can be a segment of DNAcharacterized by one or more alleles arranged along the segment, forexample, a haplotype can comprise one member of the pair of alleles foreach genetic variation or locus. In some embodiments, the haplotype cancomprise two or more alleles, three or more alleles, four or morealleles, five or more alleles, or any combination thereof, wherein, eachallele can comprise one or more genetic variations along the segment.

In some embodiments, a genetic variation can be a functional aberrationthat can alter gene function, gene expression, protein expression,protein function, or any combination thereof. In some embodiments, agenetic variation can be a loss-of-function mutation, gain-of-functionmutation, dominant negative mutation, or reversion. In some embodiments,a genetic variation can be part of a gene's coding region or regulatoryregions. Regulatory regions can control gene expression and thus proteinexpression. In some embodiments, a regulatory region can be a segment ofDNA wherein regulatory proteins, for example, transcription factors, canbind. In some embodiments a regulatory region can be positioned near thegene being regulated, for example, positions upstream of the gene beingregulated. In some embodiments, a regulatory region (e.g., enhancerelement) can be several thousands of base pairs upstream or downstreamof a gene.

In some embodiments, variants can include changes that affect apolypeptide or protein, such as a change in expression level, sequence,function, localization, binding partners, or any combination thereof. Insome embodiments, a genetic variation can be a frameshift mutation,nonsense mutation, missense mutation, neutral mutation, or silentmutation. For example, sequence differences, when compared to areference nucleotide sequence, can include the insertion or deletion ofa single nucleotide, or of more than one nucleotide, resulting in aframe shift; the change of at least one nucleotide, resulting in achange in the encoded amino acid; the change of at least one nucleotide,resulting in the generation of a premature stop codon; the deletion ofseveral nucleotides, resulting in a deletion of one or more amino acidsencoded by the nucleotides; the insertion of one or several nucleotides,such as by unequal recombination or gene conversion, resulting in aninterruption of the coding sequence of a reading frame; duplication ofall or a part of a sequence; transposition; or a rearrangement of anucleotide sequence. Such sequence changes can alter the polypeptideencoded by the nucleic acid, for example, if the change in the nucleicacid sequence causes a frame shift, the frame shift can result in achange in the encoded amino acids, and/or can result in the generationof a premature stop codon, causing generation of a truncatedpolypeptide. In some embodiments, a genetic variation associated with ENcan be a synonymous change in one or more nucleotides, for example, achange that does not result in a change in the amino acid sequence. Sucha polymorphism can, for example, alter splice sites, affect thestability or transport of mRNA, or otherwise affect the transcription ortranslation of an encoded polypeptide. In some embodiments, a synonymousmutation can result in the protein product having an altered structuredue to rare codon usage that impacts protein folding during translation,which in some cases may alter its function and/or drug bindingproperties if it is a drug target. In some embodiments, the changes thatcan alter DNA increase the possibility that structural changes, such asamplifications or deletions, occur at the somatic level. A polypeptideencoded by the reference nucleotide sequence can be a referencepolypeptide with a particular reference amino acid sequence, andpolypeptides encoded by variant nucleotide sequences can be variantpolypeptides with variant amino acid sequences.

In some embodiments, one or more variant polypeptides or proteins can beassociated with EN. In some embodiments, variant polypeptides andchanges in expression, localization, and interaction partners thereof,can be used to associate an inherited phenotype, EN, with a responsiblegenotype. In some embodiments, an EN associated variant polypeptide canbe statistically associated with a diagnosis, prognosis, or theranosisof EN.

The most common sequence variants comprise base variations at a singlebase position in the genome, and such sequence variants, orpolymorphisms, are commonly called single nucleotide polymorphisms(SNPs) or single nucleotide variants (SNVs). In some embodiments, a SNPrepresents a genetic variant present at greater than or equal to 1%occurrence in a population and in some embodiments a SNP can represent agenetic variant present at any frequency level in a population. A SNPcan be a nucleotide sequence variation occurring when a singlenucleotide at a location in the genome differs between members of aspecies or between paired chromosomes in a subject. SNPs can includevariants of a single nucleotide, for example, at a given nucleotideposition, some subjects can have a ‘G’, while others can have a ‘C’.SNPs can occur in a single mutational event, and therefore there can betwo possible alleles possible at each SNP site; the original allele andthe mutated allele. SNPs that are found to have two different bases in asingle nucleotide position are referred to as biallelic SNPs, those withthree are referred to as triallelic, and those with all four basesrepresented in the population are quadallelic. In some embodiments, SNPscan be considered neutral. In some embodiments SNPs can affectsusceptibility to EN. SNP polymorphisms can have two alleles, forexample, a subject can be homozygous for one allele of the polymorphismwherein both chromosomal copies of the individual have the samenucleotide at the SNP location, or a subject can be heterozygous whereinthe two sister chromosomes of the subject contain different nucleotides.The SNP nomenclature as reported herein is be the official Reference SNP(rs) ID identification tag as assigned to each unique SNP by theNational Center for Biotechnological Information (NCBI).

Another genetic variation of the disclosure can be copy numbervariations/variants (CNVs). CNVs can be alterations of the DNA of agenome that results in an abnormal number of copies of one or moresections of DNA. CNVs can be inherited or caused by de novo mutation andcan be responsible for a substantial amount of human phenotypicvariability, behavioral traits, and disease susceptibility. In oneembodiment, CNVs of the current disclosure can be associated with riskof or susceptibility to EN. In some embodiments, CNVs can impact asingle gene or include a contiguous set of genes. In some embodiments,CNVs can be caused by structural rearrangements of the genome, forexample, translocations, insertions, deletions, amplifications,inversions, and interstitial deletions. In some embodiments, thesestructural rearrangements occur on one or more chromosomes. Low copyrepeats (LCRs), which are region-specific repeat sequences, can besusceptible to these structural rearrangements, resulting in CNVs.Factors such as size, orientation, percentage similarity and thedistance between the copies can influence the susceptibility of LCRs tomediate genomic rearrangement.

CNVs can account for genetic variation affecting a substantialproportion of the human genome, for example, known CNVs can cover over15% of the human genome sequence (Estivill and Armengol, supra). CNVscan affect gene expression, phenotypic variation and adaptation bydisrupting a gene or altering gene dosage, and can cause disease, forexample, microdeletion and microduplication disorders, and can confersusceptibility to diseases and disorders. Updated information about thelocation, type, and size of known CNVs can be found in one or moredatabases, for example, the Database of Genomic Variants(projects.tcag.ca/variation/), which currently contains data for over100,000 CNVs.

Other types of sequence variants can be found in the human genome andcan be associated with a disease or disorder, including but not limitedto, microsatellites. Microsatellite markers are stable, polymorphic,easily analyzed, and can occur regularly throughout the genome, makingthem especially suitable for genetic analysis. A polymorphicmicrosatellite can comprise multiple small repeats of bases, forexample, CA repeats, at a particular site wherein the number of repeatlengths varies in a population. In some embodiments, microsatellites,for example, variable number of tandem repeats (VNTRs), can be shortsegments of DNA that have one or more repeated sequences, for example,about 2 to 5 nucleotides long, that can occur in non-coding DNA. In someembodiments, changes in microsatellites can occur during geneticrecombination of sexual reproduction, increasing or decreasing thenumber of repeats found at an allele, or changing allele length.

Subjects

A subject, as used herein, can be an individual of any age from whom asample containing nucleotides is obtained for analysis, e.g., by one ormore methods described herein, so as to obtain genetic data, forexample, a female adult, child, newborn, or fetus. In some embodiments,a subject can be any target of therapeutic administration. In someembodiments, a subject can be a test subject or a reference subject. Insome embodiments, a subject can be associated with EN, asymptomatic orsymptomatic, have increased or decreased susceptibility to EN, beassociated or unassociated with a treatment or treatment regimen, or anycombination thereof. As used in the present disclosure a cohort canrepresent an ethnic group, a patient group, a particular age group, agroup not associated with EN, a group associated with EN, a group ofasymptomatic female subjects, a group of symptomatic female subjects, ora group or subgroup of female subjects associated with a particularresponse to a treatment regimen or clinical trial. In some embodiments,a patient can be a subject afflicted with EN. In some embodiments, apatient can be a subject not afflicted with EN. In some embodiments, afemale subject can be a test female subject, a female patient or afemale candidate for a therapeutic, wherein genomic DNA from the femalesubject, female patient, or female candidate is obtained for analysis byone or more methods of the present disclosure herein, so as to obtaingenetic variation information of the subject, patient or candidate.

In some embodiments, the sample can be obtained prenatally from a femalefetus or embryo or from the mother, for example, from fetal or embryoniccells in the maternal circulation. In some embodiments, the sample canbe obtained with the assistance of a health care provider, for example,to draw blood. In some embodiments, the sample can be obtained withoutthe assistance of a health care provider, for example, where the sampleis obtained non-invasively, such as a saliva sample, or a samplecomprising buccal cells that is obtained using a buccal swab or brush,or a mouthwash sample.

The present disclosure also provides methods for assessing geneticvariations in female subjects who are members of a target population.Such a target population is in some embodiments a population or group ofsubjects at risk of developing EN, based on, for example, other geneticfactors, biomarkers, biophysical parameters, family history of EN,previous screening or medical history, or any combination thereof.

In some embodiments, female subjects can be from specific age subgroups,such as those over the age of 1, over the age of 2, over the age of 3,over the age of 4, over the age of 5, over the age of 6, over the age of7, over the age of 8, over the age of 9, over the age of 10, over theage of 15, over the age of 20, over the age of 25, over the age of 30,over the age of 35, over the age of 40, over the age of 45, over the ageof 50, over the age of 55, over the age of 60, over the age of 65, overthe age of 70, over the age of 75, over the age of 80, or over the ageof 85. Other embodiments of the disclosure pertain to other age groups,such as subjects aged less than 85, such as less than age 80, less thanage 75, less than age 70, less than age 65, less than age 60, less thanage 55, less than age 50, less than age 45, less than age 40, less thanage 35, less than age 30, less than age 25, less than age 20, less thanage 15, less than age 10, less than age 9, less than age 8, less thanage 6, less than age 5, less than age 4, less than age 3, less than age2, or less than age 1. Other embodiments relate to female subjects withage at onset of the disease in any of particular age or age rangesdefined by the numerical values described in the above or othernumerical values bridging these numbers. It is also contemplated that arange of ages can be relevant in certain embodiments, such as age atonset at more than age 15 but less than age 20. Other age ranges arehowever also contemplated, including all age ranges bracketed by the agevalues listed in the above.

The genetic variations of the present disclosure found to be associatedwith EN can show similar association in other female populations.Particular embodiments comprising subject female populations are thusalso contemplated and within the scope of the disclosure. Suchembodiments relate to female subjects that are from one or more humanpopulations including, but not limited to, Caucasian, European,American, Ashkenazi Jewish, Sephardi Jewish, Eurasian, Asian,Central/South Asian, East Asian, Middle Eastern, African, Hispanic, andOceanic populations. European populations include, but are not limitedto, Swedish, Norwegian, Finnish, Russian, Danish, Icelandic, Irish,Kelt, English, Scottish, Dutch, Belgian, French, German, Spanish,Portuguese, Italian, Polish, Bulgarian, Slavic, Serbian, Bosnian, Czech,Greek and Turkish populations. The racial contribution in femalesubjects can also be determined by genetic analysis, for example,genetic analysis of ancestry can be carried out using unlinkedmicrosatellite markers such as those set out in Smith et al. (Am. J.Hum. Genet., 74:1001 (2004)).

It is also well known to the person skilled in the art that certaingenetic variations have different population frequencies in differentpopulations, or are polymorphic in one population but not in another. Aperson skilled in the art can however apply the methods available and asthought herein to practice the present disclosure in any given humanpopulation. This can include assessment of genetic variations of thepresent disclosure, so as to identify those markers that give strongestassociation within the specific population. Thus, the at-risk variantsof the present disclosure can reside on different haplotype backgroundand in different frequencies in various human populations.

Samples

Samples that are suitable for use in the methods described herein can befrom a subject and can contain genetic or proteinaceous material, forexample, genomic DNA (gDNA). Genetic material can be extracted from oneor more biological samples including but not limited to, blood, saliva,urine, mucosal scrapings of the lining of the mouth, expectorant, serum,tears, skin, tissue, or hair.

In some embodiments, the sample can comprise cells or tissue, forexample, cell lines. Exemplary cell types from which genetic materialcan be obtained using the methods described herein and include but arenot limited to, a blood cell; such as a B lymphocyte, T lymphocyte,leukocyte, erythrocyte, macrophage, or neutrophil; a muscle cell such asa skeletal cell, smooth muscle cell or cardiac muscle cell; a germ cell,such as a sperm or egg; an epithelial cell; a connective tissue cell,such as an adipocyte, chondrocyte; fibroblast or osteoblast; a neuron;an astrocyte; a stromal cell; an organ specific cell, such as a kidneycell, pancreatic cell, liver cell, or a keratinocyte; a stem cell; orany cell that develops there from. A cell from which gDNA is obtainedcan be at a particular developmental level including, for example, ahematopoietic stem cell or a cell that arises from a hematopoietic stemcell such as a red blood cell, B lymphocyte, T lymphocyte, naturalkiller cell, neutrophil, basophil, eosinophil, monocyte, macrophage, orplatelet. Generally any type of stem cell can be used including, withoutlimitation, an embryonic stem cell, adult stem cell, an inducedpluripotent stem cell created from an adult cell type such asfibroblasts derived from skin or pluripotent stem cell.

In some embodiments, a sample can be processed for DNA isolation, forexample, DNA in a cell or tissue sample can be separated from othercomponents of the sample. Cells can be harvested from a biologicalsample using standard techniques known in the art, for example, bycentrifuging a cell sample and resuspending the pelleted cells, forexample, in a buffered solution, for example, phosphate-buffered saline(PBS). In some embodiments, after centrifuging the cell suspension toobtain a cell pellet, the cells can be lysed to extract DNA. In someembodiments, the sample can be concentrated and/or purified to isolateDNA. All samples obtained from a female subject, including thosesubjected to any sort of further processing, are considered to beobtained from the subject. In some embodiments, standard techniques andkits known in the art can be used to extract genomic DNA from abiological sample, including, for example, phenol extraction, a QIAamp®Tissue Kit (Qiagen, Chatsworth, Calif.), a Wizard® Genomic DNApurification kit (Promega), or a Qiagen Autopure method using Puregenechemistry, which can enable purification of highly stable DNAwell-suited for archiving.

In some embodiments, determining the identity of an allele ordetermining copy number can, but need not, include obtaining a samplecomprising DNA from a subject, and/or assessing the identity, copynumber, presence or absence of one or more genetic variations and theirchromosomal locations in the sample. The individual or organization thatperforms the determination need not actually carry out the physicalanalysis of a sample from a subject. In some embodiments, the methodscan include using information obtained by analysis of the sample by athird party. In some embodiments, the methods can include steps thatoccur at more than one site. For example, a sample can be obtained froma subject at a first site, such as at a health care provider or at thesubject's home in the case of a self-testing kit. The sample can beanalyzed at the same or a second site, for example, at a laboratory orother testing facility.

Methods of Screening

As used herein, screening a subject may include diagnosing ordetermining, theranosing, or determining the risk of or susceptibilityto developing (prognosing) EN. In particular embodiments, the disclosureis a method of determining the presence of, a risk of developing or asusceptibility to, EN, by detecting at least one genetic variation in asample from a subject as described herein. In some embodiments,detection of particular alleles, markers, variations, or haplotypes isindicative of the presence of or susceptibility to EN.

Within any given population, there can be an absolute susceptibility ofdeveloping a disease or trait, defined as the chance of a persondeveloping the specific disease or trait over a specified time-period.Susceptibility (e.g., being at-risk) is typically measured by looking atvery large numbers of people, rather than at a particular individual. Asdescribed herein, certain copy number variations (genetic variations)are found to be useful for susceptibility assessment of EN.Susceptibility assessment can involve detecting particular geneticvariations in the genome of individuals undergoing assessment.Particular genetic variations are found more frequently in individualswith EN, than in individuals without EN. Therefore, these geneticvariations have predictive value for detecting EN, risk of developingEN, or a susceptibility to EN, in an individual. Without intending to belimited by theory, it is believed that the genetic variations describedherein to be associated with susceptibility of EN represent functionalvariants predisposing to the disease. In some embodiments, a geneticvariation can confer a susceptibility of the condition, for example,carriers of the genetic variation are at a different risk of thecondition than non-carriers. In one embodiment, the presence of agenetic variation is indicative of increased susceptibility to or thepresence of EN.

Screening can be performed using any method. In some embodiments,screening can be performed using Polymerase Chain Reaction (PCR). In oneembodiment, screening can be performed using Array Comparative GenomicHybridization (aCGH). In some embodiments, the genetic variationinformation as it relates to the current disclosure can be used inconjunction with any symptomatic screening tests.

In some embodiments, information from any of the above screening methods(e.g., specific symptoms or genetic variation data) can be used todefine a subject as a test subject or reference subject. In someembodiments, information from any of the above screening methods can beused to associate a subject with a test or reference population, forexample, a subject in a population.

In one embodiment, an association with EN can be determined by thestatistical likelihood of the presence of a genetic variation in asubject with EN, for example, an unrelated individual or a first orsecond-degree relation of the subject. In some embodiments, anassociation with EN can be determined by determining the statisticallikelihood of the absence of a genetic variation in an unaffectedreference subject, for example, an unrelated individual or a first orsecond-degree relation of the subject. The methods described herein caninclude obtaining and analyzing a sample from one or more suitablereference subjects.

As used herein, susceptibility can be proneness of a subject towards thedevelopment of EN, or towards resisting development of EN, than one ormore control subjects. In some embodiments, susceptibility can encompassincreased susceptibility. For example, particular nucleic acidvariations of the disclosure as described herein can be characteristicof increased susceptibility to development of EN. In some embodiments,susceptibility can encompass decreased susceptibility, for example,particular nucleic variations of the disclosure as described herein canbe characteristic of decreased susceptibility to development of EN. Asused herein, a subject at risk of developing EN has a greater chance ofdeveloping EN relative to the general population or to one or moresubjects without a specific genetic variation.

As described herein, a genetic variation predictive of susceptibility toor presence of EN can be one where the particular genetic variation ismore frequently present in a subject with the condition (affected),compared to the frequency of its presence in a reference group(control), such that the presence of the genetic variation is indicativeof susceptibility to or presence of EN. In some embodiments, thereference group can be a population sample, for example, a random samplefrom the general population or a mixture of two or more samples from apopulation. In some embodiments, disease-free controls can becharacterized by the absence of one or more specific EN-associatedsymptoms, for example, individuals who have not experienced symptomsassociated with EN. In some embodiments, the disease-free control groupis characterized by the absence of one or more EN-specific risk factors,for example, at least one genetic and/or environmental risk factor. Insome embodiments, a reference sequence can be referred to for aparticular site of genetic variation. In some embodiments, a referenceallele can be a wild-type allele and can be chosen as either the firstsequenced allele or as the allele from a control individual. In someembodiments, one or more reference subjects can be characteristicallymatched with one or more affected subjects, for example, with matchedaged, gender or ethnicity.

A person skilled in the art can appreciate that for genetic variationswith two or more alleles present in the population being studied, andwherein one allele can found in increased frequency in a group ofindividuals with EN in the population, compared with controls, the otherallele(s) of the marker can be found in decreased frequency in the groupof individuals with the trait or disease, compared with controls. Insuch a case, one allele of the marker, for example, the allele found inincreased frequency in individuals with EN, can be the at-risk allele,while the other allele(s) can be neutral or even protective.

A genetic variant associated with EN can be used to predict thesusceptibility of EN for a given genotype. For any genetic variation,there can be one or more possible genotypes, for example, homozygote forthe at-risk variant (e.g., in autosomal recessive disorders),heterozygote, and non-carrier of the at-risk variant. In someembodiments, susceptibility associated with variants at multiple locican be used to estimate overall susceptibility. For multiple geneticvariants, there can be k (k=3{circumflex over ( )}n*2{circumflex over( )}P) possible genotypes; wherein n can be the number of autosomal lociand p can be the number of gonosomal (sex chromosomal) loci. Overallsusceptibility assessment calculations can assume that the relativesusceptibilities of different genetic variants multiply, for example,the overall susceptibility associated with a particular genotypecombination can be the product of the susceptibility values for thegenotype at each locus. If the susceptibility presented is the relativesusceptibility for a person, or a specific genotype for a person,compared to a reference population, then the combined susceptibility canbe the product of the locus specific susceptibility values and cancorrespond to an overall susceptibility estimate compared with apopulation. If the susceptibility for a person is based on a comparisonto non-carriers of the at-risk allele, then the combined susceptibilitycan correspond to an estimate that compares the person with a givencombination of genotypes at all loci to a group of individuals who donot carry at-risk variants at any of those loci. The group ofnon-carriers of any at-risk variant can have the lowest estimatedsusceptibility and can have a combined susceptibility, compared withitself, for example, non-carriers, of 1.0, but can have an overallsusceptibility, compared with the population, of less than 1.0.

Overall risk for multiple risk variants can be performed using standardmethodology. Genetic variations described herein can form the basis ofrisk analysis that combines other genetic variations known to increaserisk of EN, or other genetic risk variants for EN. In certainembodiments of the disclosure, a plurality of variants (geneticvariations, variant alleles, and/or haplotypes) can be used for overallrisk assessment. These variants are in some embodiments selected fromthe genetic variations as disclosed herein. Other embodiments includethe use of the variants of the present disclosure in combination withother variants known to be useful for screening for EN or asusceptibility to EN. In such embodiments, the genotype status of aplurality of genetic variations, markers and/or haplotypes is determinedin an individual, and the status of the individual compared with thepopulation frequency of the associated variants, or the frequency of thevariants in clinically healthy subjects, such as age-matched andsex-matched subjects.

Methods known in the art, such as the use of available algorithms andsoftware can be used to identify, or call, significant geneticvariations, including but not limited to, algorithms of DNA Analytics orDNAcopy, iPattern and/or QuantiSNP. In some embodiments, a threshold logratio value can be used to determine losses and gains. For example,using DNA Analytics, a log 2 ratio cutoff of 0.25 and −0.25 to classifyCNV gains and losses respectively may be used. As a further example,using DNAcopy, a log 2 ratio cutoff of 0.35 and −0.35 to classify CNVgains and losses respectively may be used. In some embodiments, theinformation and calls from two or more of the methods described hereincan be compared to each other to identify significant genetic variationsmore or less stringently. For example, CNV calls generated by both DNAAnalytics and DNAcopy algorithms may be defined as stringent CNVs. Insome embodiments, significant or stringent genetic variations can betagged as identified or called if it can be found to have a minimalreciprocal overlap to a genetic variation detected by one or moreplatforms and/or methods described herein. For example, a minimum of 50%reciprocal overlap can be used to tag the CNVs as identified or called.

In some embodiments, multivariate analyses or joint risk analyses,including the use of multiplicative model for overall risk assessment,and can subsequently be used to determine the overall risk conferredbased on the genotype status at the multiple loci. Use of amultiplicative model, for example, assuming that the risk of individualrisk variants multiply to establish the overall effect, allows for astraight-forward calculation of the overall risk for multiple markers.The multiplicative model is a parsimonious model that usually fits thedata of complex traits reasonably well. Deviations from multiplicityhave been rarely described in the context of common variants for commondiseases, and if reported are usually only suggestive since very largesample sizes can be required to be able to demonstrate statisticalinteractions between loci. Assessment of risk based on such analysis cansubsequently be used in the methods, uses and kits of the disclosure, asdescribed herein.

In some embodiments, the significance of increased or decreasedsusceptibility can be measured by a percentage. In some embodiments, asignificant increased susceptibility can be measured as a relativesusceptibility of at least 1.2, including but not limited to: at least1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least1.7, 1.8, at least 1.9, at least 2.0, at least 2.5, at least 3.0, atleast 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, atleast 9.0, at least 10.0, and at least 15.0. In some embodiments, arelative susceptibility of at least 2.0, at least 3.0, at least 4.0, atleast, 5.0, at least 6.0, or at least 10.0 is significant. Other valuesfor significant susceptibility are also contemplated, for example, atleast 2.5, 3.5, 4.5, 5.5, or any suitable other numerical values,wherein the values are also within scope of the present disclosure. Insome embodiments, a significant increase in susceptibility is at leastabout 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%,300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, and 1500%. In oneparticular embodiment, a significant increase in susceptibility is atleast 100%. In other embodiments, a significant increase insusceptibility is at least 200%, at least 300%, at least 400%, at least500%, at least 700%, at least 800%, at least 900% and at least 1000%.Other cutoffs or ranges as deemed suitable by the person skilled in theart to characterize the disclosure are also contemplated, and those arealso within scope of the present disclosure. In certain embodiments, asignificant increase in susceptibility is characterized by a p-value,such as a p-value of less than 0.5, less than 0.4, less than 0.3, lessthan 0.2, less than 0.1, less than 0.05, less than 0.01, less than0.001, less than 0.0001, less than 0.00001, less than 0.000001, lessthan 0.0000001, less than 0.00000001, or less than 0.000000001.

In some embodiments, an individual who is at a decreased susceptibilityfor or the lack of presence of EN can be an individual in whom at leastone genetic variation, conferring decreased susceptibility for or thelack of presence of EN is identified. In some embodiments, the geneticvariations conferring decreased susceptibility are also protective. Inone aspect, the genetic variations can confer a significant decreasedsusceptibility of or lack of presence of EN.

In some embodiments, significant decreased susceptibility can bemeasured as a relative susceptibility of less than 0.9, including butnot limited to less than 0.9, less than 0.8, less than 0.7, less than0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 and lessthan 0.1. In some embodiments, the decrease in susceptibility is atleast 20%, including but not limited to at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% and at least 98%. Other cutoffs orranges as deemed suitable by the person skilled in the art tocharacterize the disclosure are however also contemplated, and those arealso within scope of the present disclosure. In certain embodiments, asignificant decrease in susceptibility is characterized by a p-value,such as a p-value of less than 0.05, less than 0.01, less than 0.001,less than 0.0001, less than 0.00001, less than 0.000001, less than0.0000001, less than 0.00000001, or less than 0.000000001. Other testsfor significance can be used, for example, a Fisher's exact test. Otherstatistical tests of significance known to the skilled person are alsocontemplated and are also within scope of the disclosure.

In some embodiments, the significance of increased or decreasedsusceptibility can be determined according to the ratio of measurementsfrom a test subject to a reference subject. In one embodiment, losses orgains of one or more CNVs can be determined according to a thresholdlog₂ ratio determined by these measurements. In some embodiments, a log₂ratio value greater than 0.35 is indicative of a gain of one or moreCNVs. In some embodiments, a log₂ ratio value less than −0.35 isindicative of a loss of one or more CNVs.

In some embodiments, the combined or overall susceptibility associatedwith a plurality of variants associated with EN can also be assessed,for example, the genetic variations described herein to be associatedwith susceptibility to EN can be combined with other common genetic riskfactors. Combined risk for such genetic variants can be estimated in ananalogous fashion to the methods described herein.

Calculating risk conferred by a particular genotype for the individualcan be based on comparing the genotype of the individual to previouslydetermined risk expressed, for example, as a relative risk (RR) or anodds ratio (OR), for the genotype, for example, for a heterozygouscarrier of an at-risk variant for EN. An odds ratio can be a statisticalmeasure used as a metric of causality. For example, in genetic diseaseresearch it can be used to convey the significance of a variant in adisease cohort relative to an unaffected/normal cohort. The calculatedrisk for the individual can be the relative risk for a subject, or for aspecific genotype of a subject, compared to the average population. Theaverage population risk can be expressed as a weighted average of therisks of different genotypes, using results from a reference population,and the appropriate calculations to calculate the risk of a genotypegroup relative to the population can then be performed. Alternatively,the risk for an individual can be based on a comparison of particulargenotypes, for example, heterozygous carriers of an at-risk allele of amarker compared with non-carriers of the at-risk allele. Using thepopulation average can, in certain embodiments, be more convenient,since it provides a measure which can be easy to interpret for the user,for example, a measure that gives the risk for the individual, based onhis/her genotype, compared with the average in the population.

In certain embodiments of the disclosure, a genetic variation iscorrelated to EN by referencing genetic variation data to a look-uptable that comprises correlations between the genetic variation and EN.The genetic variation in certain embodiments comprises at least oneindication of the genetic variation. In some embodiments, the tablecomprises a correlation for one genetic variation. In other embodiments,the table comprises a correlation for a plurality of genetic variations.In both scenarios, by referencing to a look-up table that gives anindication of a correlation between a genetic variation and EN, a riskfor EN, or a susceptibility to EN, can be identified in the individualfrom whom the sample is derived.

The screening applications of EN-associated genetic variations, asdescribed herein, can, for example, be performed by an individual, ahealth professional, or a third party, for example, a service providerwho interprets genotype information from the subject.

A medical professional can initiate or modify treatment after receivinginformation regarding a subject's screening for EN, for example. In someembodiments, a medical professional can recommend a change in therapy.In some embodiments, a medical professional can enroll a subject in aclinical trial for, by way of example, detecting correlations between ahaplotype as described herein and any measurable or quantifiableparameter relating to the outcome of the treatment as described above.

Also provided herein are databases that include a list of geneticvariations as described herein, and wherein the list can be largely orentirely limited to genetic variations identified as useful forscreening EN as described herein. The list can be stored, for example,on a flat file or computer-readable medium. The databases can furtherinclude information regarding one or more subjects, for example, whethera subject is affected or unaffected, clinical information such asendophenotype, age of onset of symptoms, any treatments administered andoutcomes, for example, data relevant to pharmacogenomics, diagnostics,prognostics or theranostics, and other details, for example, data aboutthe disorder in the subject, or environmental or other genetic factors.The databases can be used to detect correlations between a particularhaplotype and the information regarding the subject.

The methods described herein can also include the generation of reportsfor use, for example, by a subject, care giver, or researcher, thatinclude information regarding a subject's genetic variations, andoptionally further information such as treatments administered,treatment history, medical history, predicted response, and actualresponse. The reports can be recorded in a tangible medium, e.g., acomputer-readable disk, a solid state memory device, or an opticalstorage device.

Methods of Screening Using Variations in Polypeptides and/or RNA

In some embodiments of the disclosure, screening of EN can be made byexamining or comparing changes in expression, localization, bindingpartners, and composition of a polypeptide encoded by a nucleic acidassociated with EN, for example, in those instances where the geneticvariations of the present disclosure results in a change in thecomposition or expression of the polypeptide and/or RNA, for example,mRNAs, miRNAs, and other noncoding RNAs (ncRNAs). Thus, screening of ENcan be made by examining expression and/or composition of one of thesepolypeptides and/or RNA, or another polypeptide and/or RNA encoded by anucleic acid associated with EN, in those instances where the geneticvariation of the present disclosure results in a change in theexpression, localization, binding partners, and/or composition of thepolypeptide and/or RNA. In some embodiments, screening can comprisediagnosing a subject. In some embodiments, screening can comprisedetermining a prognosis of a subject, for example, determining thesusceptibility of developing EN. In some embodiments, screening cancomprise theranosing a subject.

The genetic variations described herein that show association to EN canplay a role through their effect on one or more of these nearby genes.For example, while not intending to be limited by theory, it isgenerally expected that a deletion of a chromosomal segment comprising aparticular gene, or a fragment of a gene, can either result in analtered composition or expression, or both, of the encoded proteinand/or mRNA. Likewise, duplications, or high number copy numbervariations, are in general expected to result in increased expression ofencoded polypeptide and/or RNA if the duplication encompasses the wholegene. It is also known to those skilled in the art that segments of DNAcan be duplicated, triplicated, quadruplicated, or amplified many timesand result in increasingly higher levels of expression of the gene if itis encompassed by these multiplicated segments of DNA. Those skilled inthe art also know that one or both breakpoints of a duplication or otherlevel of amplification can disrupt a gene and thus result in loss offunction, such as the expressed protein encoded by the transcript istruncated. Further, those skilled in the art anticipate that anamplified segment of DNA can occur in tandem (e.g., multiple gene copiesadjacent to each other on the chromosome) or can insert into a site faraway from the original chromosomal location or even on anotherchromosome. Thus, in some cases a gene not contained within theamplified segment of DNA is impacted by the chromosomal rearrangement.Such complex rearrangements can be mapped, for example, by fluorescencein situ hybridization (FISH) methods. Other possible mechanismsaffecting genes within or near a genetic variation region include, forexample, effects on transcription, effects on RNA splicing, alterationsin relative amounts of alternative splice forms of mRNA, effects on RNAstability, effects on transport from the nucleus to cytoplasm, andeffects on the efficiency and accuracy of translation. Thus, DNAvariations can be detected directly, using the subjects unamplified oramplified genomic DNA, or indirectly, using RNA or DNA obtained from thesubject's tissue(s) that are present in an aberrant form or expressionlevel as a result of the genetic variations of the disclosure showingassociation to EN.

In some embodiments, the genetic variations of the disclosure showingassociation to EN can affect the expression of a gene within the geneticvariation region. Certain genetic variation regions can have flankingduplicated segments, and genes within such segments can have alteredexpression and/or composition as a result of such genomic alterations.It is also well known that regulatory elements affecting gene expressioncan be located far away, even as far as tens or hundreds of kilobasesaway, from the promoter region of a gene. Thus, regulatory elements forgenes that are located outside the genetic variation region can belocated within the genetic variation, and thus affect the expression ofgenes located outside the genetic variation. It is thus contemplatedthat the detection of the genetic variations described herein, can beused for assessing expression for one or more of associated genes.

In some embodiments, genetic variations of the disclosure showingassociation to EN can affect protein expression at the translationallevel. It can be appreciated by those skilled in the art that this canoccur by increased or decreased expression of one or more microRNAs(miRNAs) that regulates expression of a protein known to be important,or implicated, in the cause, onset, or progression of EN. Increased ordecreased expression of the one or more miRNAs can result from gain orloss of the whole miRNA gene, disruption of a portion of the gene (e.g.,by an indel or CNV), or even a single base change (SNP or SNV) thatproduces an altered, non-functional or aberrant functioning miRNAsequence. It can also be appreciated by those skilled in the art thatthe expression of protein, for example, one known to cause EN byincreased or decreased expression, can result due to a genetic variationthat results in alteration of an existing miRNA binding site within theprotein's mRNA transcript, or even creates a new miRNA binding site thatleads to aberrant protein expression.

A variety of methods can be used for detecting protein compositionand/or expression levels, including but not limited to enzyme linkedimmunosorbent assays (ELISA), Western blots, spectroscopy, massspectrometry, peptide arrays, colorimetry, electrophoresis, isoelectricfocusing, immunoprecipitations, immunoassays, and immunofluorescence andother methods well-known in the art. A test sample from a subject can beassessed for the presence of an alteration in the expression and/or analteration in composition of the polypeptide encoded by a nucleic acidassociated with EN. An “alteration” in the polypeptide expression orcomposition, as used herein, refers to an alteration in expression orcomposition in a test sample, as compared to the expression orcomposition of the polypeptide in a control sample. Such alteration can,for example, be an alteration in the quantitative polypeptide expressionor can be an alteration in the qualitative polypeptide expression, forexample, expression of a mutant polypeptide or of a different splicingvariant, or a combination thereof. In some embodiments, screening for ENcan be made by detecting a particular splicing variant encoded by anucleic acid associated with EN, or a particular pattern of splicingvariants.

Antibodies can be polyclonal or monoclonal and can be labeled orunlabeled. An intact antibody or a fragment thereof can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling adetectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled as previously described herein. Othernon-limiting examples of indirect labeling include detection of aprimary antibody using a labeled secondary antibody, for example, afluorescently-labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently-labeledstreptavidin.

Detecting Genetic Variations Associated with Endometriosis

Described herein, are methods that can be used to detect geneticvariations. Detecting specific genetic variations, for example,polymorphic markers and/or haplotypes, copy number, absence or presenceof an allele, or genotype associated with EN as described herein, can beaccomplished by methods known in the art for analyzing nucleic acidsand/or detecting sequences at polymorphic or genetically variable sites,for example, amplification techniques, hybridization techniques,sequencing, arrays, or any combination thereof. Thus, by use of thesemethods disclosed herein or other methods available to the personskilled in the art, one or more alleles at polymorphic markers,including microsatellites, SNPs, CNVs, or other types of geneticvariations, can be identified in a sample obtained from a subject.

Nucleic Acids

The nucleic acids and polypeptides described herein can be used inmethods and kits of the present disclosure. In some embodiments,aptamers that specifically bind the nucleic acids and polypeptidesdescribed herein can be used in methods and kits of the presentdisclosure. As used herein, a nucleic acid can comprise adeoxyribonucleotide (DNA) or ribonucleotide (RNA), whether singular orin polymers, naturally occurring or non-naturally occurring,double-stranded or single-stranded, coding, for example, a translatedgene, or non-coding, for example, a regulatory region, or any fragments,derivatives, mimetics or complements thereof. In some embodiments,nucleic acids can comprise oligonucleotides, nucleotides,polynucleotides, nucleic acid sequences, genomic sequences, antisensenucleic acids, DNA regions, probes, primers, genes, regulatory regions,introns, exons, open-reading frames, binding sites, target nucleic acidsand allele-specific nucleic acids.

“Isolated” nucleic acids, as used herein, are separated from nucleicacids that normally flank the gene or nucleotide sequence (as in genomicsequences) and/or has been completely or partially purified from othertranscribed sequences (e.g., as in an RNA library). For example,isolated nucleic acids of the disclosure can be substantially isolatedwith respect to the complex cellular milieu in which it naturallyoccurs, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized. Insome instances, the isolated material can form part of a composition,for example, a crude extract containing other substances, buffer systemor reagent mix. In some embodiments, the material can be purified toessential homogeneity using methods known in the art, for example, bypolyacrylamide gel electrophoresis (PAGE) or column chromatography(e.g., HPLC). With regard to genomic DNA (gDNA), the term “isolated”also can refer to nucleic acids that are separated from the chromosomewith which the genomic DNA is naturally associated. For example, theisolated nucleic acid molecule can contain less than about 250 kb, 200kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb,1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acidmolecule in the gDNA of the cell from which the nucleic acid molecule isderived.

Nucleic acids can be fused to other coding or regulatory sequences canbe considered isolated. For example, recombinant DNA contained in avector is included in the definition of “isolated” as used herein. Insome embodiments, isolated nucleic acids can include recombinant DNAmolecules in heterologous host cells or heterologous organisms, as wellas partially or substantially purified DNA molecules in solution.Isolated nucleic acids also encompass in vivo and in vitro RNAtranscripts of the DNA molecules of the present disclosure. An isolatednucleic acid molecule or nucleotide sequence can be synthesizedchemically or by recombinant means. Such isolated nucleotide sequencescan be useful, for example, in the manufacture of the encodedpolypeptide, as probes for isolating homologous sequences (e.g., fromother mammalian species), for gene mapping (e.g., by in situhybridization with chromosomes), or for detecting expression of thegene, in tissue (e.g., human tissue), such as by Northern blot analysisor other hybridization techniques disclosed herein. The disclosure alsopertains to nucleic acid sequences that hybridize under high stringencyhybridization conditions, such as for selective hybridization, to anucleotide sequence described herein Such nucleic acid sequences can bedetected and/or isolated by allele- or sequence-specific hybridization(e.g., under high stringency conditions). Stringency conditions andmethods for nucleic acid hybridizations are well known to the skilledperson (see, e.g., Current Protocols in Molecular Biology, Ausubel, F.et al., John Wiley & Sons, (1998), and Kraus, M. and Aaronson, S.,Methods Enzymol, 200:546-556 (1991), the entire teachings of which areincorporated by reference herein.

Calculations of “identity” or “percent identity” between two or morenucleotide or amino acid sequences can be determined by aligning thesequences for optimal comparison purposes (e.g., gaps can be introducedin the sequence of a first sequence). The nucleotides at correspondingpositions are then compared, and the percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions×100). For example, a position in the first sequence isoccupied by the same nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

In some embodiments, the length of a sequence aligned for comparisonpurposes is at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95%, of the length ofthe reference sequence. The actual comparison of the two sequences canbe accomplished by well-known methods, for example, using a mathematicalalgorithm. A non-limiting example of such a mathematical algorithm isdescribed in Karlin, and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873(1993). Such an algorithm is incorporated into the NBLAST and XBLASTprograms (version 2.0), as described in Altschul et al., Nucleic AcidsRes., 25:3389 (1997). When utilizing BLAST and Gapped BLAST programs,any relevant parameters of the respective programs (e.g., NBLAST) can beused. For example, parameters for sequence comparison can be set atscore=100, word length=12, or can be varied (e.g., W=5 or W=20). Otherexamples include the algorithm of Myers and Miller, CABIOS (1989),ADVANCE, ADAM, BLAT, and FASTA. In some embodiments, the percentidentity between two amino acid sequences can be accomplished using, forexample, the GAP program in the GCG software package (Accelrys,Cambridge, UK).

“Probes” or “primers” can be oligonucleotides that hybridize in abase-specific manner to a complementary strand of a nucleic acidmolecule. Probes can include primers, which can be a single-strandedoligonucleotide probe that can act as a point of initiation oftemplate-directed DNA synthesis using methods including but not limitedto, polymerase chain reaction (PCR) and ligase chain reaction (LCR) foramplification of a target sequence. It can be appreciated by thoseskilled in the art that probes for detection of amplified or unamplifiednucleic acid molecules can also include an Invader oligonucleotide andprobe pair. Oligonucleotides, as described herein, can include segmentsor fragments of nucleic acid sequences, or their complements. In someembodiments, DNA segments can be between 5 and 10,000 contiguous bases,and can range from 5, 10, 12, 15, 20, or 25 nucleotides to 10, 15, 20,25, 30, 40, 50, 100, 200, 500, 1000 or 10,000 nucleotides. In additionto DNA and RNA, probes and primers can include polypeptide nucleic acids(PNA), as described in Nielsen, P. et al., Science 254: 1497-1500(1991). A probe or primer can comprise a region of nucleotide sequencethat hybridizes to at least about 15, typically about 20-25, and incertain embodiments about 40, 50 or 75, consecutive nucleotides of anucleic acid molecule.

The present disclosure also provides isolated nucleic acids, forexample, probes or primers, that contain a fragment or portion that canselectively hybridize to a nucleic acid that comprises, or consists of,a nucleotide sequence, wherein the nucleotide sequence can comprise atleast one polymorphism or polymorphic allele contained in the geneticvariations described herein or the wild-type nucleotide that is locatedat the same position, or the compliments thereof. In some embodiments,the probe or primer can be at least 70% identical, at least 80%identical, at least 85% identical, at least 90% identical, or at least95% identical, to the contiguous nucleotide sequence or to thecomplement of the contiguous nucleotide sequence.

In one embodiment, a nucleic acid probe can be an oligonucleotidecapable of hybridizing with a complementary region of a gene associatedwith EN containing a genetic variation described herein. The nucleicacid fragments of the disclosure can be used as probes or primers inassays such as those described herein.

The nucleic acids of the disclosure, such as those described above, canbe identified and isolated using standard molecular biology techniqueswell known to the skilled person. In some embodiments, DNA can beamplified and/or can be labeled (e.g., radiolabeled, fluorescentlylabeled) and used as a probe for screening, for example, a cDNA libraryderived from an organism. cDNA can be derived from mRNA and can becontained in a suitable vector. For example, corresponding clones can beisolated, DNA obtained fallowing in vivo excision, and the cloned insertcan be sequenced in either or both orientations by art-recognizedmethods to identify the correct reading frame encoding a polypeptide ofthe appropriate molecular weight. Using these or similar methods, thepolypeptide and the DNA encoding the polypeptide can be isolated,sequenced and further characterized.

In some embodiments, nucleic acid can comprise one or morepolymorphisms, variations, or mutations, for example, single nucleotidepolymorphisms (SNPs), copy number variations (CNVs), for example,insertions, deletions, inversions, and translocations. In someembodiments, nucleic acids can comprise analogs, for example,phosphorothioates, phosphoramidates, methyl phosphonate, chiral methylphosphonates, 2-O-methyl ribonucleotides, or modified nucleic acids, forexample, modified backbone residues or linkages, or nucleic acidscombined with carbohydrates, lipids, protein or other materials, orpeptide nucleic acids (PNAs), for example, chromatin, ribosomes, andtranscriptosomes. In some embodiments nucleic acids can comprise nucleicacids in various structures, for example, A DNA, B DNA, Z-form DNA,siRNA, tRNA, and ribozymes. In some embodiments, the nucleic acid may benaturally or non-naturally polymorphic, for example, having one or moresequence differences, for example, additions, deletions and/orsubstitutions, as compared to a reference sequence. In some embodiments,a reference sequence can be based on publicly available information, forexample, the U.C. Santa Cruz Human Genome Browser Gateway(genome.ucsc.edu/cgi-bin/hgGateway) or the NCBI website(www.ncbi.nlm.nih.gov). In some embodiments, a reference sequence can bedetermined by a practitioner of the present disclosure using methodswell known in the art, for example, by sequencing a reference nucleicacid.

In some embodiments, a probe can hybridize to an allele, SNP, or CNV asdescribed herein. In some embodiments, the probe can bind to anothermarker sequence associated with EN as described herein.

One of skill in the art would know how to design a probe so thatsequence specific hybridization can occur only if a particular allele ispresent in a genomic sequence from a test sample. The disclosure canalso be reduced to practice using any convenient genotyping method,including commercially available technologies and methods for genotypingparticular genetic variations

Control probes can also be used, for example, a probe that binds a lessvariable sequence, for example, a repetitive DNA associated with acentromere of a chromosome, can be used as a control. In someembodiments, probes can be obtained from commercial sources. In someembodiments, probes can be synthesized, for example, chemically or invitro, or made from chromosomal or genomic DNA through standardtechniques. In some embodiments sources of DNA that can be used includegenomic DNA, cloned DNA sequences, somatic cell hybrids that containone, or a part of one, human chromosome along with the normal chromosomecomplement of the host, and chromosomes purified by flow cytometry ormicrodissection. The region of interest can be isolated through cloning,or by site-specific amplification using PCR.

One or more nucleic acids for example, a probe or primer, can also belabeled, for example, by direct labeling, to comprise a detectablelabel. A detectable label can comprise any label capable of detection bya physical, chemical, or a biological process for example, a radioactivelabel, such as ³²P or ³H, a fluorescent label, such as FITC, achromophore label, an affinity-ligand label, an enzyme label, such asalkaline phosphatase, horseradish peroxidase, or 12 galactosidase, anenzyme cofactor label, a hapten conjugate label, such as digoxigenin ordinitrophenyl, a Raman signal generating label, a magnetic label, a spinlabel, an epitope label, such as the FLAG or HA epitope, a luminescentlabel, a heavy atom label, a nanoparticle label, an electrochemicallabel, a light scattering label, a spherical shell label, semiconductornanocrystal label, such as quantum dots (described in U.S. Pat. No.6,207,392), and probes labeled with any other signal generating labelknown to those of skill in the art, wherein a label can allow the probeto be visualized with or without a secondary detection molecule. Anucleotide can be directly incorporated into a probe with standardtechniques, for example, nick translation, random priming, and PCRlabeling.

Non-limiting examples of label moieties useful for detection include,without limitation, suitable enzymes such as horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;members of a binding pair that are capable of forming complexes such asstreptavidin/biotin, avidin/biotin or an antigen/antibody complexincluding, for example, rabbit IgG and anti-rabbit IgG; fluorophoressuch as umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, tetramethyl rhodamine, eosin, green fluorescent protein,erythrosin, coumarin, methyl coumarin, pyrene, malachite green,stilbene, lucifer yellow, Cascade Blue, Texas Red,dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin,fluorescent lanthanide complexes such as those including Europium andTerbium, cyanine dye family members, such as Cy3 and Cy5, molecularbeacons and fluorescent derivatives thereof, as well as others known inthe art as described, for example, in Principles of FluorescenceSpectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition(July 1999) and the 6th Edition of the Molecular Probes Handbook byRichard P. Hoagland; a luminescent material such as luminol; lightscattering or plasmon resonant materials such as gold or silverparticles or quantum dots; or radioactive material include ¹⁴C, ¹²³I,¹²⁴I, ¹²⁵I, Tc99m, ³²P, ³³P, ³⁵S or ³H.

Other labels can also be used in the methods of the present disclosure,for example, backbone labels. Backbone labels comprise nucleic acidstains that bind nucleic acids in a sequence independent manner.Non-limiting examples include intercalating dyes such as phenanthridinesand acridines (e.g., ethidium bromide, propidium iodide, hexidiumiodide, dihydroethidium, ethidium homodimer-1 and -2, ethidiummonoazide, and ACMA); some minor grove binders such as indoles andimidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI);and miscellaneous nucleic acid stains such as acridine orange (alsocapable of intercalating), 7-AAD, actinomycin D, LDS751, andhydroxystilbamidine. All of the aforementioned nucleic acid stains arecommercially available from suppliers such as Molecular Probes, Inc.Still other examples of nucleic acid stains include the following dyesfrom Molecular Probes: cyanine dyes such as SYTOX Blue, SYTOX Green,SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1,LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3,TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3,PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II,SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24,-21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82,-83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red).

In some embodiments, fluorophores of different colors can be chosen, forexample, 7-amino-4-methylcoumarin-3-acetic acid (AMCA),5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B,5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC),7-diethylaminocoumarin-3-carboxylic acid,tetramethylrhodamine-5-(and-6)-isothiocyanate,5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylicacid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionicacid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, TRITC,rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7,Texas Red, Phar-Red, allophycocyanin (APC), and CASCADE blueacetylazide, such that each probe in or not in a set can be distinctlyvisualized. In some embodiments, fluorescently labeled probes can beviewed with a fluorescence microscope and an appropriate filter for eachfluorophore, or by using dual or triple band-pass filter sets to observemultiple fluorophores. In some embodiments, techniques such as flowcytometry can be used to examine the hybridization pattern of theprobes.

In other embodiments, the probes can be indirectly labeled, for example,with biotin or digoxygenin, or labeled with radioactive isotopes such as³²P and/or ³H. As a non-limiting example, a probe indirectly labeledwith biotin can be detected by avidin conjugated to a detectable marker.For example, avidin can be conjugated to an enzymatic marker such asalkaline phosphatase or horseradish peroxidase. In some embodiments,enzymatic markers can be detected using colorimetric reactions using asubstrate and/or a catalyst for the enzyme. In some embodiments,catalysts for alkaline phosphatase can be used, for example,5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium. In someembodiments, a catalyst can be used for horseradish peroxidase, forexample, diaminobenzoate.

Methods of Detecting Genetic Variations

In some embodiments, standard techniques for genotyping for the presencegenetic variations, for example, amplification, can be used.Amplification of nucleic acids can be accomplished using methods knownin the art. Generally, sequence information from the region of interestcan be used to design oligonucleotide primers that can be identical orsimilar in sequence to opposite strands of a template to be amplified.In some embodiments, amplification methods can include but are notlimited to, fluorescence-based techniques utilizing PCR, for example,ligase chain reaction (LCR), Nested PCR, transcription amplification,self-sustained sequence replication, nucleic acid based sequenceamplification (NASBA), and multiplex ligation-dependent probeamplification (MLPA). Guidelines for selecting primers for PCRamplification are well known in the art. In some embodiments, a computerprogram can be used to design primers, for example, Oligo (NationalBiosciences, Inc., Plymouth Minn.), MacVector (Kodak/IBI), and GCG suiteof sequence analysis programs.

In some embodiments, commercial methodologies available for genotyping,for example, SNP genotyping, can be used, but are not limited to, TaqMangenotyping assays (Applied Biosystems), SNPlex platforms (AppliedBiosystems), gel electrophoresis, capillary electrophoresis, sizeexclusion chromatography, mass spectrometry, for example, MassARRAYsystem (Sequenom), minisequencing methods, real-time Polymerase ChainReaction (PCR), Bio-Plex system (BioRad), CEQ and SNPstream systems(Beckman), array hybridization technology, for example, AffymetrixGeneChip (Perlegen), BeadArray Technologies, for example, IlluminaGoldenGate and Infinium assays, array tag technology, MultiplexLigation-dependent Probe Amplification (MLPA), and endonuclease-basedfluorescence hybridization technology (Invader; Third Wave/Hologic). PCRcan be a procedure in which target nucleic acid is amplified in a mannersimilar to that described in U.S. Pat. No. 4,683,195 and subsequentmodifications of the procedure described therein. In some embodiments,real-time quantitative PCR can be used to determine genetic variations,wherein quantitative PCR can permit both detection and quantification ofa DNA sequence in a sample, for example, as an absolute number of copiesor as a relative amount when normalized to DNA input or othernormalizing genes. In some embodiments, methods of quantification caninclude the use of fluorescent dyes that can intercalate withdouble-stranded DNA, and modified DNA oligonucleotide probes that canfluoresce when hybridized with a complementary DNA.

In some embodiments of the disclosure, a sample containing genomic DNAobtained from the subject can be collected and PCR can used to amplify afragment of nucleic acid that comprises one or more genetic variationsthat can be indicative of a susceptibility to EN. In some embodiments,detection of genetic variations can be accomplished by expressionanalysis, for example, by using quantitative PCR. In some embodiments,this technique can assess the presence of an alteration in theexpression or composition of one or more polypeptides or splicingvariants encoded by a nucleic acid associated with EN.

In one embodiment, the DNA template of a sample from a subjectcontaining a SNP can be amplified by PCR prior to detection with aprobe. In such an embodiment, the amplified DNA serves as the templatefor a detection probe and, in some embodiments, an enhancer probe.Certain embodiments of the detection probe, the enhancer probe, and/orthe primers used for amplification of the template by PCR can comprisethe use of modified bases, for example, modified A, T, C, G, and U,wherein the use of modified bases can be useful for adjusting themelting temperature of the nucleotide probe and/or primer to thetemplate DNA. In one embodiment, modified bases are used in the designof the detection nucleotide probe. Any modified base known to theskilled person can be selected in these methods, and the selection ofsuitable bases is well within the scope of the skilled person based onthe teachings herein and known bases available from commercial sourcesas known to the skilled person.

In some embodiments, identification of genetic variations can beaccomplished using hybridization methods. The presence of a specificmarker allele or a particular genomic segment comprising a geneticvariation, or representative of a genetic variation, can be indicated bysequence-specific hybridization of a nucleic acid probe specific for theparticular allele or the genetic variation in a nucleic acid containingsample that has or has not been amplified but methods described herein.The presence of more than one specific marker allele or several geneticvariations can be indicated by using two or more sequence-specificnucleic acid probes, wherein each is specific for a particular alleleand/or genetic variation.

Hybridization can be performed by methods well known to the personskilled in the art, for example, hybridization techniques such asfluorescent in situ hybridization (FISH), Southern analysis, Northernanalysis, or in situ hybridization. In some embodiments, hybridizationrefers to specific hybridization, wherein hybridization can be performedwith no mismatches. Specific hybridization, if present, can be usingstandard methods. In some embodiments, if specific hybridization occursbetween a nucleic acid probe and the nucleic acid in the sample, thesample can contain a sequence that can be complementary to a nucleotidepresent in the nucleic acid probe. In some embodiments, if a nucleicacid probe can contain a particular allele of a polymorphic marker, orparticular alleles for a plurality of markers, specific hybridization isindicative of the nucleic acid being completely complementary to thenucleic acid probe, including the particular alleles at polymorphicmarkers within the probe. In some embodiments a probe can contain morethan one marker allele of a particular haplotype, for example, a probecan contain alleles complementary to 2, 3, 4, 5 or all of the markersthat make up a particular haplotype. In some embodiments detection ofone or more particular markers of the haplotype in the sample isindicative that the source of the sample has the particular haplotype.

In some embodiments, PCR conditions and primers can be developed thatamplify a product only when the variant allele is present or only whenthe wild type allele is present, for example, allele-specific PCR. Insome embodiments of allele-specific PCR, a method utilizing a detectionoligonucleotide probe comprising a fluorescent moiety or group at its 3′terminus and a quencher at its 5′ terminus, and an enhanceroligonucleotide, can be employed, as described by Kutyavin et al.(Nucleic Acid Res., 34:e128 (2006)).

An allele-specific primer/probe can be an oligonucleotide that isspecific for particular a polymorphism can be prepared using standardmethods. In some embodiments, allele-specific oligonucleotide probes canspecifically hybridize to a nucleic acid region that contains a geneticvariation. In some embodiments, hybridization conditions can be selectedsuch that a nucleic acid probe can specifically bind to the sequence ofinterest, for example, the variant nucleic acid sequence.

In some embodiments, allele-specific restriction digest analysis can beused to detect the existence of a polymorphic variant of a polymorphism,if alternate polymorphic variants of the polymorphism can result in thecreation or elimination of a restriction site. Allele-specificrestriction digests can be performed, for example, with the particularrestriction enzyme that can differentiate the alleles. In someembodiments, PCR can be used to amplify a region comprising thepolymorphic site, and restriction fragment length polymorphism analysiscan be conducted. In some embodiments, for sequence variants that do notalter a common restriction site, mutagenic primers can be designed thatcan introduce one or more restriction sites when the variant allele ispresent or when the wild type allele is present.

In some embodiments, fluorescence polarization template-directeddye-terminator incorporation (FP-TDI) can be used to determine which ofmultiple polymorphic variants of a polymorphism can be present in asubject. Unlike the use of allele-specific probes or primers, thismethod can employ primers that can terminate adjacent to a polymorphicsite, so that extension of the primer by a single nucleotide can resultin incorporation of a nucleotide complementary to the polymorphicvariant at the polymorphic site.

In some embodiments, DNA containing an amplified portion can bedot-blotted, using standard methods and the blot contacted with theoligonucleotide probe. The presence of specific hybridization of theprobe to the DNA can then be detected. The methods can includedetermining the genotype of a subject with respect to both copies of thepolymorphic site present in the genome, wherein if multiple polymorphicvariants exist at a site, this can be appropriately indicated byspecifying which variants are present in a subject. Any of the detectionmeans described herein can be used to determine the genotype of asubject with respect to one or both copies of the polymorphism presentin the subject's genome.

In some embodiments, a peptide nucleic acid (PNA) probe can be used inaddition to, or instead of, a nucleic acid probe in the methodsdescribed herein. A PNA can be a DNA mimic having a peptide-like,inorganic backbone, for example, N-(2-aminoethyl) glycine units with anorganic base (A, G, C, T or U) attached to the glycine nitrogen via amethylene carbonyl linker.

Nucleic acid sequence analysis can also be used to detect geneticvariations, for example, genetic variations can be detected bysequencing exons, introns, 5′ untranslated sequences, or 3′ untranslatedsequences. One or more methods of nucleic acid analysis that areavailable to those skilled in the art can be used to detect geneticvariations, including but not limited to, direct manual sequencing,automated fluorescent sequencing, single-stranded conformationpolymorphism assays (SSCP); clamped denaturing gel electrophoresis(CDGE); denaturing gradient gel electrophoresis (DGGE), two-dimensionalgel electrophoresis (2DGE or TDGE); conformational sensitive gelelectrophoresis (CSGE); denaturing high performance liquidchromatography (DHPLC), infrared matrix-assisted laserdesorption/ionization (IR-MALDI) mass spectrometry, mobility shiftanalysis, quantitative real-time PCR, restriction enzyme analysis,heteroduplex analysis; chemical mismatch cleavage (CMC), RNaseprotection assays, use of polypeptides that recognize nucleotidemismatches, allele-specific PCR, real-time pyrophosphate DNA sequencing,PCR amplification in combination with denaturing high performance liquidchromatography (dHPLC), and combinations of such methods.

Sequencing can be accomplished through classic Sanger sequencingmethods, which are known in the art. In one embodiment sequencing can beperformed using high-throughput sequencing methods some of which allowdetection of a sequenced nucleotide immediately after or upon itsincorporation into a growing strand, for example, detection of sequencein substantially real time or real time. In some cases, high throughputsequencing generates at least 1,000, at least 5,000, at least 10,000, atleast 20,000, at least 30,000, at least 40,000, at least 50,000, atleast 100,000 or at least 500,000 sequence reads per hour; with eachread being at least 50, at least 60, at least 70, at least 80, at least90, at least 100, at least 120 or at least 150 bases per read (or500-1,000 bases per read for 454).

High-throughput sequencing methods can include but are not limited to,Massively Parallel Signature Sequencing (MPSS, Lynx Therapeutics),Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing,SOLiD sequencing, on semiconductor sequencing, DNA nanoball sequencing,Helioscope™ single molecule sequencing, Single Molecule SMRT sequencing,Single Molecule real time (RNAP) sequencing, Nanopore DNA sequencing,and/or sequencing by hybridization, for example, a non-enzymatic methodthat uses a DNA microarray, or microfluidic Sanger sequencing.

In some embodiments, high-throughput sequencing can involve the use oftechnology available by Helicos BioSciences Corporation (Cambridge,Mass.) such as the Single Molecule Sequencing by Synthesis (SMSS)method. SMSS is unique because it allows for sequencing the entire humangenome in up to 24 hours. This fast sequencing method also allows fordetection of a SNP/nucleotide in a sequence in substantially real timeor real time. Finally, SMSS is powerful because, like the MIPtechnology, it does not use a pre-amplification step prior tohybridization. SMSS does not use any amplification. SMSS is described inUS Publication Application Nos. 20060024711; 20060024678; 20060012793;20060012784; and 20050100932. In some embodiments, high-throughputsequencing involves the use of technology available by 454 LifeSciences, Inc. (a Roche company, Branford, Conn.) such as thePicoTiterPlate device which includes a fiber optic plate that transmitschemiluminescent signal generated by the sequencing reaction to berecorded by a CCD camera in the instrument. This use of fiber opticsallows for the detection of a minimum of 20 million base pairs in 4.5hours.

In some embodiments, PCR-amplified single-strand nucleic acid can behybridized to a primer and incubated with a polymerase, ATP sulfurylase,luciferase, apyrase, and the substrates luciferin and adenosine 5′phosphosulfate. Next, deoxynucleotide triphosphates corresponding to thebases A, C, G, and T (U) can be added sequentially. A base incorporationcan be accompanied by release of pyrophosphate, which can be convertedto ATP by sulfurylase, which can drive synthesis of oxyluciferin and therelease of visible light. Since pyrophosphate release can be equimolarwith the number of incorporated bases, the light given off can beproportional to the number of nucleotides adding in any one step. Theprocess can repeat until the entire sequence can be determined. In someembodiments, pyrosequencing can be utilized to analyze amplicons todetermine whether breakpoints are present. In some embodiments,pyrosequencing can map surrounding sequences as an internal qualitycontrol.

Pyrosequencing analysis methods are known in the art. Sequence analysiscan include a four-color sequencing by ligation scheme (degenerateligation), which involves hybridizing an anchor primer to one of fourpositions. Then an enzymatic ligation reaction of the anchor primer to apopulation of degenerate nonamers that are labeled with fluorescent dyescan be performed. At any given cycle, the population of nonamers that isused can be structured such that the identity of one of its positionscan be correlated with the identity of the fluorophore attached to thatnonamer. To the extent that the ligase discriminates for complementarilyat that queried position, the fluorescent signal can allow the inferenceof the identity of the base. After performing the ligation andfour-color imaging, the anchor primer: nonamer complexes can be strippedand a new cycle begins. Methods to image sequence information afterperforming ligation are known in the art.

In some embodiments, analysis by restriction enzyme digestion can beused to detect a particular genetic variation if the genetic variationresults in creation or elimination of one or more restriction sitesrelative to a reference sequence. In some embodiments, restrictionfragment length polymorphism (RFLP) analysis can be conducted, whereinthe digestion pattern of the relevant DNA fragment indicates thepresence or absence of the particular genetic variation in the sample.

In some embodiments, arrays of oligonucleotide probes that can becomplementary to target nucleic acid sequence segments from a subjectcan be used to identify genetic variations. In some embodiments, anarray of oligonucleotide probes comprises an oligonucleotide array, forexample, a microarray. In some embodiments, the present disclosurefeatures arrays that include a substrate having a plurality ofaddressable areas, and methods of using them. At least one area of theplurality includes a nucleic acid probe that binds specifically to asequence comprising a genetic variation, and can be used to detect theabsence or presence of the genetic variation, for example, one or moreSNPs, microsatellites, or CNVs, as described herein, to determine oridentify an allele or genotype. For example, the array can include oneor more nucleic acid probes that can be used to detect a geneticvariation associated with a gene and/or product of a gene listed in FIG.3 . In some embodiments, the array can further comprise at least onearea that includes a nucleic acid probe that can be used to specificallydetect another marker associated with EN, as described herein.

Microarray hybridization can be performed by hybridizing a nucleic acidof interest, for example, a nucleic acid encompassing a geneticvariation, with the array and detecting hybridization using nucleic acidprobes. In some embodiments, the nucleic acid of interest is amplifiedprior to hybridization. Hybridization and detecting can be carried outaccording to standard methods described in Published PCT Applications:WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186. For example,an array can be scanned to determine the position on the array to whichthe nucleic acid hybridizes. The hybridization data obtained from thescan can be, for example, in the form of fluorescence intensities as afunction of location on the array.

Arrays can be formed on substrates fabricated with materials such aspaper; glass; plastic, for example, polypropylene, nylon, orpolystyrene; polyacrylamide; nitrocellulose; silicon; optical fiber; orany other suitable solid or semisolid support; and can be configured ina planar, for example, glass plates or silicon chips); or threedimensional, for example, pins, fibers, beads, particles, microtiterwells, and capillaries, configuration.

Methods for generating arrays are known in the art and can include forexample; photolithographic methods (U.S. Pat. Nos. 5,143,854, 5,510,270and 5,527,681); mechanical methods, for example, directed-flow methods(U.S. Pat. No. 5,384,261); pin-based methods (U.S. Pat. No. 5,288,514);bead-based techniques (PCT US/93/04145); solid phase oligonucleotidesynthesis methods; or by other methods known to a person skilled in theart (see, e.g., Bier et al., Adv. Biochem. Eng. Biotechnol., 109:433-53(2008); Hoheisel, Nat. Rev. Genet., 7: 200-10 (2006); Fan et al.,Methods Enzymol., 410:57-73 (2006); Raqoussis & Elvidge, Expert Rev.Mol. Design, 6: 145-52 (2006); Mockler et al., Genomics, 85:1-15 (2005),and references cited therein, the entire teachings of each of which areincorporated by reference herein). Many additional descriptions of thepreparation and use of oligonucleotide arrays for detection ofpolymorphisms can be found, for example, in U.S. Pat. Nos. 6,858,394;6,429,027; 5,445,934; 5,700,637; 5,744,305; 5,945,334; 6,054,270;6,300,063; 6,733,977; 7,364,858, EP 619 321, and EP 373 203, the entireteachings of which are incorporated by reference herein. Methods forarray production, hybridization, and analysis are also described inSnijders et al., Nat. Genetics, 29:263-264 (2001); Klein et al., Proc.Natl. Acad. Sci. USA, 96:4494-4499 (1999); Albertson et al., BreastCancer Research and Treatment, 78:289-298 (2003); and Snijders et al.,“BAC microarray based comparative genomic hybridization,” in: Zhao etal. (eds), Bacterial Artificial Chromosomes: Methods and Protocols,Methods in Molecular Biology, Humana Press, 2002.

In some embodiments, oligonucleotide probes forming an array can beattached to a substrate by any number of techniques, including, but notlimited to, in situ synthesis, for example, high-density oligonucleotidearrays, using photolithographic techniques; spotting/printing a mediumto low density on glass, nylon, or nitrocellulose; by masking; and bydot-blotting on a nylon or nitrocellulose hybridization membrane. Insome embodiments, oligonucleotides can be immobilized via a linker,including but not limited to, by covalent, ionic, or physical linkage.Linkers for immobilizing nucleic acids and polypeptides, includingreversible or cleavable linkers, are known in the art (U.S. Pat. No.5,451,683 and WO98/20019). In some embodiments, oligonucleotides can benon-covalently immobilized on a substrate by hybridization to anchors,by means of magnetic beads, or in a fluid phase, for example, in wellsor capillaries.

An array can comprise oligonucleotide hybridization probes capable ofspecifically hybridizing to different genetic variations. In someembodiments, oligonucleotide arrays can comprise a plurality ofdifferent oligonucleotide probes coupled to a surface of a substrate indifferent known locations. In some embodiments, oligonucleotide probescan exhibit differential or selective binding to polymorphic sites, andcan be readily be designed by one of ordinary skill in the art, forexample, an oligonucleotide that is perfectly complementary to asequence that encompasses a polymorphic site, for example, a sequencethat includes the polymorphic site, within it, or at one end, canhybridize preferentially to a nucleic acid comprising that sequence, asopposed to a nucleic acid comprising an alternate polymorphic variant.

In some embodiments, arrays can include multiple detection blocks, forexample, multiple groups of probes designed for detection of particularpolymorphisms. In some embodiments, these arrays can be used to analyzemultiple different polymorphisms. In some embodiments, detection blockscan be grouped within a single array or in multiple, separate arrays,wherein varying conditions, for example, conditions optimized forparticular polymorphisms, can be used during hybridization. Generaldescriptions of using oligonucleotide arrays for detection ofpolymorphisms can be found, for example, in U.S. Pat. Nos. 5,858,659 and5,837,832. In addition to oligonucleotide arrays, cDNA arrays can beused similarly in certain embodiments.

The methods described herein can include but are not limited toproviding an array as described herein; contacting the array with asample, and detecting binding of a nucleic acid from the sample to thearray. In some embodiments, the method can comprise amplifying nucleicacid from the sample, for example, a region associated with EN or aregion that includes another region associated with EN. In someembodiments, the methods described herein can include using an arraythat can identify differential expression patterns or copy numbers ofone or more genes in samples from control and affected individuals. Forexample, arrays of probes to a marker described herein can be used toidentify genetic variations between DNA from an affected subject, andcontrol DNA obtained from an individual that does not have EN. Since thenucleotides on the array can contain sequence tags, their positions onthe array can be accurately known relative to the genomic sequence.

In some embodiments, it can be desirable to employ methods that candetect the presence of multiple genetic variations, for example,polymorphic variants at a plurality of polymorphic sites, in parallel orsubstantially simultaneously. In some embodiments, these methods cancomprise oligonucleotide arrays and other methods, including methods inwhich reactions, for example, amplification and hybridization, can beperformed in individual vessels, for example, within individual wells ofa multi-well plate or other vessel.

Determining the identity of a genetic variation can also include orconsist of reviewing a subject's medical history, where the medicalhistory includes information regarding the identity, copy number,presence or absence of one or more alleles or SNPs in the subject, e.g.,results of a genetic test.

Genetic variations can also be identified using any of a number ofmethods well known in the art. For example, genetic variations availablein public databases, which can be searched using methods and customalgorithms or algorithms known in the art, can be used. In someembodiments, a reference sequence can be from, for example, the humandraft genome sequence, publicly available in various databases, or asequence deposited in a database such as GenBank.

Methods of Detecting CNVs

Detection of genetic variations, specifically CNVs, can be accomplishedby one or more suitable techniques described herein. Generally,techniques that can selectively determine whether a particularchromosomal segment is present or absent in an individual can be usedfor genotyping CNVs. Identification of novel copy number variations canbe done by methods for assessing genomic copy number changes.

In some embodiments, methods include but are not limited to, methodsthat can quantitatively estimate the number of copies of a particulargenomic segment, but can also include methods that indicate whether aparticular segment is present in a sample or not. In some embodiments,the technique to be used can quantify the amount of segment present, forexample, determining whether a DNA segment is deleted, duplicated, ortriplicated in subject, for example, Fluorescent In Situ Hybridization(FISH) techniques, and other methods described herein.

In some embodiments, other genotyping technologies can be used fordetection of CNVs, including but not limited to, karyotype analysis,Molecular Inversion Probe array technology, for example, Affymetrix SNPArray 6.0, and BeadArray Technologies, for example, Illumina GoldenGateand Infinium assays, as can other platforms such as NimbleGen HD2.1 orHD4.2, High-Definition Comparative Genomic Hybridization (CGH) arrays(Agilent Technologies), tiling array technology (Affymetrix), multiplexligation-dependent probe amplification (MLPA), Invader assay, qPCR, orfluorescence in situ hybridization. In one embodiment, Array ComparativeGenomic Hybridization (aCGH) methods can be used. As described herein,karyotype analysis can be a method to determine the content andstructure of chromosomes in a sample. In some embodiments, karyotypingcan be used, in lieu of aCGH, to detect translocations, which can becopy number neutral (balanced translocations), and therefore, notdetectable by aCGH. Information about amplitude of particular probes,which can be representative of particular alleles, can providequantitative dosage information for the particular allele, and byconsequence, dosage information about the CNV in question, since themarker can be selected as a marker representative of the CNV and can belocated within the CNV. In some embodiments, if the CNV is a deletion,the absence of particular marker allele is representative of thedeletion. In some embodiments, if the CNV is a duplication or a higherorder copy number variation, the signal intensity representative of theallele correlating with the CNV can represent the copy number. A summaryof methodologies commonly used is provided in Perkel (J. Nature Methods,5:447-453 (2008)).

PCR assays can be utilized to detect CNVs and can provide an alternativeto array analysis. In particular, PCR assays can enable detection ofprecise boundaries of gene/chromosome variants, at the molecular level,and which boundaries are identical in different individuals. PCR assayscan be based on the amplification of a junction fragment present only inindividuals that carry a deletion. This assay can convert the detectionof a loss by array CGH to one of a gain by PCR.

Examples of PCR techniques that can be used in the present disclosureinclude, but are not limited to quantitative PCR, real-time quantitativePCR (qPCR), quantitative fluorescent PCR (QF-PCR), multiplex fluorescentPCR (MF-PCR), real time PCR (RT-PCR), single cell PCR,PCR-RFLP/RT-PCR-RFLP, hot start PCR and Nested PCR. Other suitableamplification methods include the ligase chain reaction (LCR), ligationmediated PCR (LM-PCR), degenerate oligonucleotide probe PCR (DOP-PCR),transcription amplification, self-sustained sequence replication,selective amplification of target polynucleotide sequences, consensussequence primed polymerase chain reaction (CP-PCR), arbitrarily primedpolymerase chain reaction (AP-PCR) and nucleic acid based sequenceamplification (NABSA).

Alternative methods for the simultaneous interrogation of multipleregions include quantitative multiplex PCR of short fluorescentfragments (QMPSF), multiplex amplifiable probe hybridization (MAPH) andmultiplex ligation-dependent probe amplification (MLPA), in whichcopy-number differences for up to 40 regions can be scored in oneexperiment. Another approach can be to specifically target regions thatharbor known segmental duplications, which are often sites ofcopy-number variation. By targeting the variable nucleotides between twocopies of a segmental duplication (called paralogous sequence variants)using a SNP-genotyping method that provides independent fluorescenceintensities for the two alleles, it is possible to detect an increase inintensity of one allele compared with the other.

In some embodiments, the amplified piece of DNA can be bound to beadsusing the sequencing element of the nucleic acid tag under conditionsthat favor a single amplified piece of DNA molecule to bind a differentbead and amplification occurs on each bead. In some embodiments, suchamplification can occur by PCR. Each bead can be placed in a separatewell, which can be a picoliter-sized well. In some embodiments, eachbead is captured within a droplet of aPCR-reaction-mixture-in-oil-emulsion and PCR amplification occurs withineach droplet. The amplification on the bead results in each beadcarrying at least one million, at least 5 million, or at least 10million copies of the single amplified piece of DNA molecule.

In some embodiments where PCR occurs in oil-emulsion mixtures, theemulsion droplets are broken, the DNA is denatured and the beadscarrying single-stranded nucleic acids clones are deposited into a well,such as a picoliter-sized well, for further analysis according to themethods described herein. These amplification methods allow for theanalysis of genomic DNA regions. Methods for using bead amplificationfollowed by fiber optics detection are described in Margulies et al.,Nature, 15:437(7057):376-80 (2005), and as well as in US PublicationApplication Nos. 20020012930; 20030068629; 20030100102; 20030148344;20040248161; 20050079510, 20050124022; and 20060078909.

Another variation on the array-based approach can be to use thehybridization signal intensities that are obtained from theoligonucleotides employed on Affymetrix SNP arrays or in Illumina BeadArrays. Here hybridization intensities are compared with average valuesthat are derived from controls, such that deviations from these averagesindicate a change in copy number. As well as providing information aboutcopy number, SNP arrays have the added advantage of providing genotypeinformation. For example, they can reveal loss of heterozygosity, whichcould provide supporting evidence for the presence of a deletion, ormight indicate segmental uniparental disomy (which can recapitulate theeffects of structural variation in some genomic regions—Prader-Willi andAngelman syndromes, for example).

Many of the basic procedures followed in microarray-based genomeprofiling are similar, if not identical, to those followed in expressionprofiling and SNP analysis, including the use of specialized microarrayequipment and data-analysis tools. Since microarray-based expressionprofiling has been well established in the last decade, much can belearned from the technical advances made in this area. Examples of theuse of microarrays in nucleic acid analysis that can be used aredescribed in U.S. Pat. Nos. 6,300,063, 5,837,832, 6,969,589, 6,040,138,6,858,412, U.S. application Ser. No. 08/529,115, U.S. application Ser.No. 10/272,384, U.S. application Ser. No. 10/045,575, U.S. applicationSer. No. 10/264,571 and U.S. application Ser. No. 10/264,574. It shouldbe noted that there are also distinct differences such as target andprobe complexity, stability of DNA over RNA, the presence of repetitiveDNA and the need to identify single copy number alterations in genomeprofiling.

In one embodiment, the genetic variations detected comprise CNVs and maybe detected using array CGH. In some embodiments, array CGH can be beenimplemented using a wide variety of techniques. The initial approachesused arrays produced from large-insert genomic clones such as bacterialartificial chromosomes (BACs). Producing sufficient BAC DNA of adequatepurity to make arrays is arduous, so several techniques to amplify smallamounts of starting material have been employed. These techniquesinclude ligation-mediated PCR (Snijders et al, Nat. Genet., 29:263-64),degenerate primer PCR using one or several sets of primers, and rollingcircle amplification. BAC arrays that provide complete genome tilingpaths are also available. Arrays made from less complex nucleic acidssuch as cDNAs, selected PCR products, and oligonucleotides can also beused. Although most CGH procedures employ hybridization with totalgenomic DNA, it is possible to use reduced complexity representations ofthe genome produced by PCR techniques. Computational analysis of thegenome sequence can be used to design array elements complementary tothe sequences contained in the representation. Various SNP genotypingplatforms, some of which use reduced complexity genomic representations,can be useful for their ability to determine both DNA copy number andallelic content across the genome. In some embodiments, small amounts ofgenomic DNA can be amplified with a variety of whole genomeamplification methods prior to CGH analysis of the sample.

The different basic approaches to array CGH provide different levels ofperformance, so some are more suitable for particular applications thanothers. The factors that determine performance include the magnitudes ofthe copy number changes, their genomic extents, the state andcomposition of the specimen, how much material is available foranalysis, and how the results of the analysis can be used. Manyapplications use reliable detection of copy number changes of much lessthan 50%, a higher stringency than for other microarray technologies.Note that technical details are extremely important and differentimplementations of methods using the same array CGH approach can yielddifferent levels of performance. Various CGH methods are known in theart and are equally applicable to one or more methods of the presentdisclosure. For example, CGH methods are disclosed in U.S. Pat. Nos.7,957,913, 7,910,353, 7,238,484, 7,702,468, 7,034,144; 7,030,231;7,011,949; 7,014,997; 6,977,148; 6,951,761; and 6,916,621, thedisclosure from each of which is incorporated by reference herein in itsentirety.

The data provided by array CGH are quantitative measures of DNA sequencedosage. Array CGH provides high-resolution estimates of copy numberaberrations, and can be performed efficiently on many samples. Theadvent of array CGH technology makes it possible to monitor DNA copynumber changes on a genomic scale and many projects have been launchedfor studying the genome in specific diseases.

In one embodiment, whole genome array-based comparative genomehybridization (array CGH) analysis, or array CGH on a subset of genomicregions, can be used to efficiently interrogate human genomes forgenomic imbalances at multiple loci within a single assay. Thedevelopment of comparative genomic hybridization (CGH) (Kallioniemi etal, 1992, Science 258: 818-21) provided the first efficient approach toscanning entire genomes for variations in DNA copy number. Theimportance of normal copy number variation involving large segments ofDNA has been unappreciated. Array CGH is a breakthrough technique inhuman genetics, which is attracting interest from clinicians working infields as diverse as cancer and IVF (In Vitro Fertilization). The use ofCGH microarrays in the clinic holds great promise for identifyingregions of genomic imbalance associated with disease. Advances fromidentifying chromosomal critical regions associated with specificphenotypes to identifying the specific dosage sensitive genes can leadto therapeutic opportunities of benefit to patients. Array CGH is aspecific, sensitive and rapid technique that can enable the screening ofthe whole genome in a single test. It can facilitate and accelerate thescreening process in human genetics and is expected to have a profoundimpact on the screening and counseling of patients with geneticdisorders. It is now possible to identify the exact location on thechromosome where an aberration has occurred and it is possible to mapthese changes directly onto the genomic sequence.

An array CGH approach provides a robust method for carrying out agenome-wide scan to find novel copy number variants (CNVs). The arrayCGH methods can use labeled fragments from a genome of interest, whichcan be competitively hybridized with a second differentially labeledgenome to arrays that are spotted with cloned DNA fragments, revealingcopy-number differences between the two genomes. Genomic clones (forexample, BACs), cDNAs, PCR products and oligonucleotides, can all beused as array targets. The use of array CGH with BACs was one of theearliest employed methods and is popular, owing to the extensivecoverage of the genome it provides, the availability of reliable mappingdata and ready access to clones. The last of these factors is importantboth for the array experiments themselves, and for confirmatory FISHexperiments.

In a typical CGH measurement, total genomic DNA is isolated from testand reference subjects, differentially labeled, and hybridized to arepresentation of the genome that allows the binding of sequences atdifferent genomic locations to be distinguished. More than two genomescan be compared simultaneously with suitable labels. Hybridization ofhighly repetitive sequences is typically suppressed by the inclusion ofunlabeled Cot-1 DNA in the reaction. The relative hybridizationintensity of the test and reference signals at a given location can beproportional to the relative copy number of those sequences in the testand reference genomes. If the reference genome is normal then increasesand decreases in signal intensity ratios directly indicate DNA copynumber variation within the test genome. Data are typically normalizedso that the modal ratio for the genome is set to some standard value,typically 1.0 on a linear scale or 0.0 on a logarithmic scale.Additional measurements such as FISH or flow cytometry can be used todetermine the actual copy number associated with a ratio level.

In some embodiments, an array CGH procedure can include the followingsteps. First, large-insert clones, for example, BACs can be obtainedfrom a supplier of clone libraries. Then, small amounts of clone DNA canbe amplified, for example, by degenerate oligonucleotide-primed (DOP)PCR or ligation-mediated PCR in order to obtain sufficient quantitiesneeded for spotting. Next, PCR products can be spotted onto glass slidesusing, for example, microarray robots equipped with high-precisionprinting pins. Depending on the number of clones to be spotted and thespace available on the microarray slide, clones can either be spottedonce per array or in replicate. Repeated spotting of the same clone onan array can increase precision of the measurements if the spotintensities are averaged, and allows for a detailed statistical analysisof the quality of the experiments. Subject and control DNAs can belabeled, for example, with either Cy3 or Cy5-dUTP using random primingand can be subsequently hybridized onto the microarray in a solutioncontaining an excess of Cot1-DNA to block repetitive sequences.Hybridizations can either be performed manually under a coverslip, in agasket with gentle rocking or, automatically using commerciallyavailable hybridization stations. These automated hybridization stationscan allow for an active hybridization process, thereby improving thereproducibility as well as reducing the actual hybridization time, whichincreases throughput. The hybridized DNAs can detected through the twodifferent fluorochromes using standard microarray scanning equipmentwith either a scanning confocal laser or a charge coupled device (CCD)camera-based reader, followed by spot identification using commerciallyor freely available software packages.

The use of CGH with arrays that comprise long oligonucleotides (60-100bp) can improve the detection resolution (in some embodiments, as smallas about 3-5 kb sized CNVs on arrays designed for interrogation of humanwhole genomes) over that achieved using BACs (limited to 50-100 kb orlarger sized CNVs due to the large size of BAC clones). In someembodiments, the resolution of oligonucleotide CGH arrays is achievedvia in situ synthesis of 1-4 million unique features/probes permicroarray, which can include microarrays available from Roche NimbleGenand Agilent Technologies. In addition to array CGH methods for copynumber detection, other embodiments for partial or whole genome analysisof CNVs within a genome include, but are not limited to, use of SNPgenotyping microarrays and sequencing methods.

Another method for copy number detection that uses oligonucleotides canbe representational oligonucleotide microarray analysis (ROMA). It issimilar to that applied in the use of BAC and CGH arrays, but toincrease the signal-to-noise ratio, the ‘complexity’ of the input DNA isreduced by a method called representation or whole-genome sampling. Herethe DNA that is to be hybridized to the array can be treated byrestriction digestion and then ligated to adapters, which results in thePCR-based amplification of fragments in a specific size-range. As aresult, the amplified DNA can make up a fraction of the entire genomicsequence—that is, it is a representation of the input DNA that hassignificantly reduced complexity, which can lead to a reduction inbackground noise. Other suitable methods available to the skilled personcan also be used, and are within scope of the present disclosure.

A comparison of one or more genomes relative to one or more othergenomes with array CGH, or a variety of other CNV detection methods, canreveal the set of CNVs between two genomes, between one genome incomparison to multiple genomes, or between one set of genomes incomparison to another set of genomes. In some embodiments, an array CGHexperiment can be performed by hybridizing a single test genome againsta pooled sample of two or more genomes, which can result in minimizingthe detection of higher frequency variants in the experiment. In someembodiments, a test genome can be hybridized alone (e.g., one-colordetection) to a microarray, for example, using array CGH or SNPgenotyping methods, and the comparison step to one or more referencegenomes can be performed in silico to reveal the set of CNVs in the testgenome relative to the one or more reference genomes. In one embodiment,a single test genome is compared to a single reference genome in a2-color experiment wherein both genomes are cohybridized to themicroarray.

Array CGH can be used to identify genes that are causative or associatedwith a particular phenotype, condition, or disease by comparing the setof CNVs found in the affected cohort to the set of CNVs found in anunaffected cohort. An unaffected cohort may consist of any individualunaffected by the phenotype, condition, or disease of interest, but inone embodiment is comprised of individuals or subjects that areapparently healthy (normal). Methods employed for such analyses aredescribed in U.S. Pat. Nos. 7,702,468, 7,957,913 and 8,862,410. In someembodiments of CNV comparison methods, candidate genes that arecausative or associated (i.e., potentially serving as a biomarker) witha phenotype, condition, or disease will be identified by CNVs that occurin the affected cohort but not in the unaffected cohort, or present atmuch lower frequency in the unaffected cohort as compared to theaffected cohort. In another embodiment of CNV comparison methods, one ormore CNVs may be present at much higher frequency in the unaffectedcohort as compared to the affected cohort and thus may be indicative ofprotection for development of the disease or condition present in theaffected cohort. In some embodiments of CNV comparison methods,candidate genes that are causative or associated (i.e., potentiallyserving as a biomarker) with a phenotype, condition, or disease will beidentified by CNVs that occur at a statistically significant higherfrequency in the affected cohort as compared their frequency in theunaffected cohort. Thus, CNVs detected in the affected cohort ascompared to the unaffected cohort can serve as beacons of genes that arecausative or associated with a particular phenotype, condition, ordisease. In some embodiments, CNV detection and comparison methods canresult in direct identification of the gene that is causative orassociated with phenotype, condition, or disease if the CNVs are foundto overlap with or encompass the gene(s). In some embodiments, CNVdetection and comparison methods can result in identification ofregulatory regions of the genome (e.g., promoters, enhancers,transcription factor binding sites) that regulate the expression of oneor more genes that are causative or associated with the phenotype,condition, or disease of interest.

Due to the large amount of genetic variation between any two genomes, ortwo sets (cohorts) of genomes, being compared, one embodiment is toreduce the genetic variation search space by interrogating only CNVs, asopposed to the full set of genetic variants that can be identified in anindividual's genome or exome. The set of CNVs that occur only, or at astatistically higher frequency, in the affected cohort as compared tothe unaffected cohort can then be further investigated in targetedsequencing experiments to reveal the full set of genetic variants (ofany size or type) that are causative or associated (e.g., potentiallyserving as a biomarker) with a phenotype, condition, or disease. It canbe appreciated by those skilled in the art that the targeted sequencingexperiments can be performed in both the affected and unaffected cohortsin order to identify the genetic variants (e.g., SNVs and indels) thatoccur only, or at a statistically significant higher frequency, in theaffected individual or cohort as compared to the unaffected cohort. Inanother embodiment, the targeted sequencing experiments can be performedon the affected cohort and the variations found can be compared topublic or private databases containing sequence variants present inunaffected subjects, or in some embodiments, the general population.

When investigating EN, it can be appreciated by those skilled in the artthat the number of EN candidate genes (or regulatory sequences)identified via CNV (or other variant types) detection methods mayincrease or decrease when additional EN cohorts are analyzed. Similarly,the number of EN candidate genes (or regulatory sequences), for example,identified via CNV (or other variant types) detection methods mayincrease or decrease when additional unaffected cohorts are used tointerpret the affected cohort CNVs (or other variant types). For veryrare CNVs (e.g., <0.1% frequency in the general population), only asingle case may be observed in a given EN cohort (e.g., 100 cases) butfurther statistical significance or evidence for the gene (or regulatorysequence/locus in the genome) can be established by: 1) CNV analysis ofadditional EN cohorts, 2) CNV analysis of additional Normal cohorts, 3)targeted gene sequencing of both EN and Normal cohorts, and/or 4)functional characterization of the EN candidate gene (e.g., in silicoanalysis of the predicted impact of the candidate mutation on the geneproduct, RNAi knockdown experiments, biochemical assays on EN patienttissue, gene expression analysis of disease-relevant tissues or ofinduced pluripotent stem cells (iPSCs) created from the EN patient(s)harboring the candidate EN-causing genetic variant). It can beappreciated by those skilled in the art that the ability to identifydisease genes via rare CNVs in as few as 100-300 cases, the typical sizeof a Phase 2 clinical trial, has particular utility for theidentification of genetic biomarkers of drug efficacy and/or safety andadvantages over SNV-based discovery methods, which typically requirethousands of cases. Genes identified by rare loss-of-function CNVs canthen be sequenced in the Phase 2 clinical trial cohort (or, via targetedinterpretation of previously obtained exome and/or whole genomesequences on the clinical trial cohort) to reveal genetic biomarkers.Knowledge of genetic biomarkers for safety and/or efficacy in Phase 2can substantially reduce the attrition rate and costs of drugdevelopment.

It can be appreciated by those skilled in the art that a candidate genemay validate as causative of the phenotype, condition, or disease (e.g.,EN), which may, for example, be confirmed via mechanism of actionexperiments, or it may serve as a biomarker of the phenotype, condition,or disease. Thus, in the example of EN, in some embodiments, theEN-specific gene (or regulatory sequence/locus) may be a biomarker ofage-of-onset for EN and disease severity, and thus have diagnosticutility for monitoring patients known to be at risk for EN or as ageneral screening test in the population for early diagnosis of thedisease. In some embodiments, the EN-specific gene/biomarker may be anindicator of drug response (e.g., a particular subtype of EN may respondbest to a therapeutic targeting a particular phenotype, causative gene,or other gene in the same pathway as the causative gene) and thus haveutility during drug development in clinical trials. For example,clinical trials for a therapeutic that targets a EN genetic subtypecomprising only 10% of all patients exhibiting symptoms of EN, can bedesigned to comprise only those 10% of patients with a specificgenotype(s) in order to reduce the time and cost of such clinical trials(e.g., smaller number of patients in the clinical trial). It can beappreciated by those skilled in the art that such patient stratificationmethods (i.e., specific genotypes correlated with the disease or drugresponse) can be employed not only for targeted therapeutics, but ingeneral for any drug that is approved or in development (i.e., themechanism of action may or may not be known). For example, drugs indevelopment or approved to treat, for example, cancer, may have utilityin being repurposed to treat EN. Such patient stratification methods canalso be utilized to develop a companion diagnostic test (e.g.,comprising the specific genes/genotypes found in patients that areindicative of drug response) for a particular drug, either concurrentlyduring the clinical trials for the drug or after drug approval (e.g., asa new indication or for the physician to use in guiding medicaldecisions for the patient).

Further links to EN pathology may be established via pathway analysis ofthe genes, which may take into consideration binding interactions (e.g.,via yeast 2-hybrid screen) and molecular events (e.g., kinase activityor other enzymatic processes) if such information is available for thegene(s) of interest (e.g., specified in the analysis). Both commercial(e.g., Ingenuity's IPA software and Thomson Reuter's GeneGo software)and open source software (e.g., String: string-db.org/) are availablefor such analyses. To assess connections to established EN biology,analyses can be performed for the set of candidate EN genesindependently or against known causative EN genes singly or as a group.For example, see FIG. 9 .

A method of screening a subject for a disease or disorder can compriseassaying a nucleic acid sample from the subject to detect sequenceinformation for more than one genetic loci and comparing the sequenceinformation to a panel of nucleic acid biomarkers and screening thesubject for the presence or absence of EN if one or more of lowfrequency biomarkers in the panel are present in the sequenceinformation. The panel may comprise at least one nucleic acid biomarkerfor each of the more than one genetic loci. For example, the panel cancomprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75,100, 150, 200 or more nucleic acid biomarkers for each of the more thanone genetic loci. The panel may comprise at least 25 low frequencybiomarkers. For example, the panel can comprise at least 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 135, 150, 175, 200, 250,500, or 1000 or more low frequency biomarkers. A low frequency biomarkercan occur at a frequency of 0.1% or less in a population of subjectswithout a diagnosis of the disease or disorder. For example, a lowfrequency biomarker can occur at a frequency of 0.05%, 0.01%, 0.005%,0.001%, 0.0005%, 0.0001%, 0.00005%, or 0.00001% or less in a populationof subjects without a diagnosis of the disease or disorder.

In some embodiments, the presence or absence of EN in the subject can bedetermined with at least 50% confidence. For example, the presence orabsence of the disease or disorder in the subject can be determined withat least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% confidence.

In one embodiment, EN candidate CNV subregions and genes associated withthese regions may be determined or identified by comparing genetic datafrom a cohort of normal individuals (NVE) to that of a cohort ofindividuals known to have, or be susceptible to EN.

In some embodiments, genomic DNA samples from individuals within a NVEcohort and/or a EN cohort can be considered test subject DNA samples andhybridized against one or more, sex-matched reference DNA samples fromindividuals. For example, reference DNA samples can be labeled with afluorophore such as Cy5, using methods described herein, and testsubject DNA samples can be labeled with a different fluorophore, such asCy3. After labeling, samples can be combined and can be co-hybridized toa microarray and analyzed using any of the methods described herein,such as aCGH. Arrays can then be scanned and the data can be analyzedwith software. Genetic alterations, such as CNVs, can be called usingany of the methods described herein. A list of the genetic alterations,such as CNVs, can be generated for each cohort. The list of CNVs can beused to generate a master list of non-redundant CNVs and/or CNVsubregions for each cohort. The list can be based on the presence orabsence of the CNV subregion in individuals within the cohort. In thismanner, the master list can contain a number of distinct CNV subregions,some of which are uniquely present in a single individual and some ofwhich are present in multiple individuals.

In some embodiments, CNV subregions of interest may be obtained byannotation of each CNV subregion with relevant information, such asoverlap with known genes and/or exons. In some embodiments, CNVsubregions of interest can be obtained by calculating the OR for a CNVsubregion according to the following formula: OR=(EN/((# individuals inEN cohort)−EN))/(NVE/((# individuals in NVE cohort)−NVE)), where:EN=number of EN individuals with a CNV subregion of interest andNVE=number of NVE individuals with the CNV subregion of interest. IfNVE=0, it can be set to 1 to avoid dealing with infinities in caseswhere no CNVs are seen in the NVE.

The number of individuals in any given cohort may be at least about 10,50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 5000, 7500,10,000, 100,000, or more.

In some embodiments, a CNV subregion/gene can be of interest if the CNVsubregion overlaps a known gene, and is associated with an OR of atleast 6, e.g., at least 35. For example, a CNV subregion/gene can be ofinterest if the CNV subregion overlaps a known gene, and is associatedwith an OR of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40,45, 50, or more. In some embodiments, a CNV subregion/gene can be ofinterest if the CNV subregion overlaps a known gene, and is associatedwith an OR from about 6-100, 6-50, 6-40, 6-30, 6-20, 6-10, 6-9, 6-8,6-7, 8-100, 8-50, 8-40, 8-30, 8-20, 8-10, 10-100, 10-50, 10-40, 10-30,10-20, 20-100, 20-50, 20-40, 20-30, 30-100, 30-50, 30-40, 40-100, 40-50,50-100, or 5-7. The CNV subregion/gene can be an exonic or intronic partof the gene, or both.

In some embodiments, a CNV subregion/gene can be of interest if the CNVsubregion does not overlap a known gene (e.g., is non-genic orintergenic) and is associated with an OR of at least 4 or higher. Forexample, a CNV subregion/gene can be of interest if the CNV subregiondoes not overlap a known gene (e.g., is non-genic or intergenic) and isassociated with an OR of at least 5, 6, 7, 9, 9, 10, 12, 14, 16, 18, 20,25, 30, 35, 40, 45, 50, or more. In some embodiments, a CNVsubregion/gene can be of interest if the CNV subregion does not overlapa known gene (e.g., is non-genic or intergenic) and is associated withan OR from about 5-100, 5-50, 5-40, 5-30, 5-20, 20-100, 20-50, 20-40,20-30, 30-100, 30-50, 30-40, 40-100, 40-50, 50-100, or 9-11.

In some embodiments, a CNV subregion/gene can be of interest based onthe OR associated with the sum of EN cases and the sum of NVE casesaffecting the same gene (including distinct CNV subregions). Forexample, a CNV subregion/gene can be of interest if the OR associatedwith the sum of EN cases and the sum of NVE cases affecting the samegene (including distinct CNV subregions) is at least 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, or more. In someembodiments, a CNV subregion/gene can be of interest if the ORassociated with the sum of EN cases and the sum of NVE cases affectingthe same gene (including distinct CNV subregions) is from about 4-100,4-50, 4-40, 4-30, 4-20, 4-10, 4-9, 4-8, 4-7, 8-100, 8-50, 8-40, 8-30,8-20, 8-10, 10-100, 10-50, 10-40, 10-30, 10-20, 20-100, 20-50, 20-40,20-30, 30-100, 30-50, 30-40, 40-100, 40-50, 50-100, or 5-7.

The data presented in FIGS. 1-4 were generated on the basis of acomparison of copy number variants (CNVs) identified in a NVE and a ENcohort. CNV genome locations are provided using the Human Mar. 2006(NCBI36/hg18) assembly. It can be appreciated by those skilled in theart that a CNV found in an affected individual may have one or moresubregions that are found in the affected cohort as compared to theunaffected cohort and, similarly, other subregions within the CNV thatare found at comparable frequencies, or not statistically significantdifferent frequencies, in the affected and unaffected cohorts. In oneembodiment, CNV detection and analysis methods are employed that enablecomparison of CNV subregions to facilitate identification of genes (orregulatory loci) that are causative or associated with the phenotype,condition, or disease being investigated (or detected for diagnosticpurposes).

FIG. 1 lists exemplary CNVs associated with EN, obtained as described inExample 1. For each entry, the chromosome (for the purpose of algorithmsand databases used in the analyses, chromosome X is designated aschromosome 23) and original CNV start and stop positions are listed,along with original CNV size, CNV type (loss or gain), EN case ID, geneannotation (for the CNV subregion not the original CNV) andcorresponding SEQ ID numbers (Nos.).

FIG. 2 shows the actual CNV subregions found to be unique orsignificantly different between the EN and NVE cohorts. For each entry,the chromosome (for the purpose of algorithms and databases used in theanalyses, chromosome X is designated as chromosome 23) and CNV subregionstart and stop positions are listed, along with CNV subregion size, CNVtype (loss or gain), EN case ID, gene annotation, whether a genic CNVsubregion of interest overlaps an exon or not, the number of NVEsubjects and the number of EN subjects that harbor the relevant CNVsubregion, the Fisher's 2 tailed Exact Test (FET), Odds ratio (OR), andthe category under which the CNV subregion was identified.

FIG. 3 represents a non-redundant list for all genes listed in FIG. 2(namely, those relevant to CNV subregions of interest), and includesRefSeq Gene Symbol, Exon overlap (intronic, exonic or both), NCBI GeneID (DNA accession number), Gene Description (brief gene description),and RefSeq Summary (summary of gene function).

FIG. 4 represents a non-redundant list for all genes listed in FIG. 2(namely, those relevant to CNV subregions of interest) and includesRefSeq Gene Symbol, Exon overlap (intronic, exonic or both), RefSeqAccession Number (may be multiple entries per gene), mRNA Description(brief description of mRNA), and corresponding SEQ ID Nos.

More than one RNA product (e.g., alternatively spliced mRNA transcriptsand non-coding RNAs) can be produced from a single gene. FIG. 4 listsall presently known transcript variants (and their RNA accessionnumbers) but new variants may be found when further studies arecompleted and that generation of these additional transcript variants(and ultimately protein and/or regulatory RNA products) may also beimpacted by one or more CNVs or CNV subregions listed in FIGS. 1 and 2 ,respectively. The transcripts listed in FIG. 4 can be expressionproducts of the same gene biomarker. The gene biomarker can comprisegenomic DNA encoding the gene, including exons, introns, and/orregulatory binding regions (such as enhancers, promoters, silencers,and/or response elements). Point mutations, polymorphisms,translocations, insertions, deletions, amplifications, inversions,microsatellites, interstitial deletions, CNVs, loss of heterozygosity,or any other aberrations which affect the structure or function of oneor more gene biomarkers and/or expression products thereof, can beassociated with EN as described herein.

Computer-Implemented Aspects

As understood by those of ordinary skill in the art, the methods andinformation described herein (genetic variation association with EN) canbe implemented, in all or in part, as computer executable instructionson known computer readable media. For example, the methods describedherein can be implemented in hardware. Alternatively, the method can beimplemented in software stored in, for example, one or more memories orother computer readable medium and implemented on one or moreprocessors. As is known, the processors can be associated with one ormore controllers, calculation units and/or other units of a computersystem, or implanted in firmware as desired. If implemented in software,the routines can be stored in any computer readable memory such as inRAM, ROM, flash memory, a magnetic disk, a laser disk, or other storagemedium, as is also known. Likewise, this software can be delivered to acomputing device via any known delivery method including, for example,over a communication channel such as a telephone line, the Internet, awireless connection, etc., or via a transportable medium, such as acomputer readable disk, flash drive, etc.

More generally, and as understood by those of ordinary skill in the art,the various steps described above can be implemented as various blocks,operations, tools, modules and techniques which, in turn, can beimplemented in hardware, firmware, software, or any combination ofhardware, firmware, and/or software. When implemented in hardware, someor all of the blocks, operations, techniques, etc. can be implementedin, for example, a custom integrated circuit (IC), an applicationspecific integrated circuit (ASIC), a field programmable logic array(FPGA), a programmable logic array (PLA), etc.

Results from such genotyping can be stored in a data storage unit, suchas a data carrier, including computer databases, data storage disks, orby other convenient data storage means. In certain embodiments, thecomputer database is an object database, a relational database or apost-relational database. Data can be retrieved from the data storageunit using any convenient data query method.

When implemented in software, the software can be stored in any knowncomputer readable medium such as on a magnetic disk, an optical disk, orother storage medium, in a RAM or ROM or flash memory of a computer,processor, hard disk drive, optical disk drive, tape drive, etc.Likewise, the software can be delivered to a user or a computing systemvia any known delivery method including, for example, on a computerreadable disk or other transportable computer storage mechanism.

The steps of the claimed methods can be operational with numerous othergeneral purpose or special purpose computing system environments orconfigurations. Examples of well-known computing systems, environments,and/or configurations that can be suitable for use with the methods orsystem of the claims include, but are not limited to, personalcomputers, server computers, hand-held or laptop devices, multiprocessorsystems, microprocessor-based systems, set top boxes, programmableconsumer electronics, network PCs, minicomputers, mainframe computers,distributed computing environments that include any of the above systemsor devices, and the like.

The steps of the claimed method and system can be described in thegeneral context of computer-executable instructions, such as programmodules, being executed by a computer. Generally, program modulesinclude routines, programs, objects, components, and/or data structuresthat perform particular tasks or implement particular abstract datatypes. The methods and apparatus can also be practiced in distributedcomputing environments where tasks are performed by remote processingdevices that are linked through a communications network. In bothintegrated and distributed computing environments, program modules canbe located in both local and remote computer storage media includingmemory storage devices. Numerous alternative embodiments could beimplemented, using either current technology or technology developedafter the filing date of this application, which would still fall withinthe scope of the claims defining the disclosure.

While the risk evaluation system and method, and other elements, havebeen described as being implemented in software, they can be implementedin hardware, firmware, etc., and can be implemented by any otherprocessor. Thus, the elements described herein can be implemented in astandard multi-purpose CPU or on specifically designed hardware orfirmware such as an application-specific integrated circuit (ASIC) orother hard-wired device as desired. When implemented in software, thesoftware routine can be stored in any computer readable memory such ason a magnetic disk, a laser disk, or other storage medium, in a RAM orROM of a computer or processor, in any database, etc. Likewise, thissoftware can be delivered to a user or a screening system via any knownor desired delivery method including, for example, on a computerreadable disk or other transportable computer storage mechanism or overa communication channel, for example a telephone line, the internet, orwireless communication. Modifications and variations can be made in thetechniques and structures described and illustrated herein withoutdeparting from the spirit and scope of the present disclosure.

EN Therapeutics

Medications and surgery can provide relief from the symptoms of EN. Themain families of drugs useful for treating EN are NSAIDS, GnRH agonistsand antagonists, oral contraceptives, progestin, danazol, and aromataseinhibitors. The stage of the disease may determine which therapy is mostuseful.

It can be appreciated by those skilled in the art that patientstratification methods (e.g., specific genotypes correlated with thedisease or drug response) can be employed not only for targetedtherapeutics, but in general for any drug that is approved or indevelopment. For example, drugs in development or approved to treat, forexample, cancer, may have utility in being repurposed to treat EN. Suchpatient stratification methods can also be utilized to develop acompanion diagnostic test (e.g., for the specific genes/genotypes foundin patients that are indicative of drug response) for a particular drug,either concurrently during the clinical trials for the drug or afterdrug approval (e.g., as a new indication or for the physician to use inguiding medical decisions for the patient).

Thus, those skilled in the art can appreciate that the gene product ofone or more EN-associated genes listed in FIGS. 1-3 may be useful as adrug target for development of a therapeutic to treat a patient with anEN-associated genetic variant. For example, the protein product of agene that is known to contain an EN-associated variant (such as an SNVor CNV) may have partial or full loss of function in an EN patient withthe EN-associated genetic variant. In another embodiment, anEN-associated genetic variant may result in gain of function for theprotein product expressed from the mutated gene, such as an activatingmutation of the protein's function (e.g., kinase activity is aberrantlyincreased) or over-expression of the protein due to a CNV gain of thewhole gene. Methods of drug screening for drug targets are well known tothose skilled in the art. Drugs can be developed to specifically targeta gene that is found to cause a particular disease (e.g., EN) due to thepresence of a genetic variant or to another gene in a particularbiochemical/cellular pathway (e.g., FIG. 9 ).

In one embodiment, drug development can be pursued on the basis that agene is a target for drugs in development or already approved for use inone or more conditions, such as those genes listed in FIG. 10 . Forexample, EN-associated genes in FIG. 3 that are known drug targets,according to FIG. 10 , include CYP17A1, LEPR, PTK2, RXFP1, and TSHR. Inanother embodiment, a gene family member of a known drug target may havea higher probability of success in yielding an approved drug than atarget with no prior efforts attempted for drug development. Forexample, EN-associated genes in FIG. 3 that are in the same gene familyof known drug targets, according to FIG. 10 , include DPP6 (FIG. 10 ,DPP4), GPR111 (FIG. 10 , 4 GPR genes), HMGB3 (FIG. 10 , HMGB1), MAGEA11(FIG. 10 , MAGEA3), MUC4 (FIG. 10 , MUC1 and MUC16), MYO1B (FIG. 10 ,MYO7A), PDE1C (FIG. 10 , 6 PDE genes), PGRMC2 (FIG. 10 , PGR), PLA2G4C(FIG. 10 , PLA2G4A), TGFBR3 (FIG. 10 , TGFBR1 and TGFBR2), and TGFB1I1(FIG. 10 , TGFB signaling genes TGFB1, TGFB2, TGFBR1, TGFBR2).

For example, a drug approved or in development for one disease area mayhave therapeutic use in a related, or even unrelated, disease area. Forexample, the EN-associated gene RXFP1 is a known drug target (FIG. 10 )and multiple pharmaceutical companies are developing drugs that targetits gene product. The RXFP1 protein is a G-protein-coupled receptor andthe endogenous hormone relaxin activates this receptor (Xiao et al.,Probe Reports from the NIH Molecular Libraries Program [Internet]Bethesda (Md.): National Center for Biotechnology Information (US),2010-2012 (2013, updated); Xiao et al., Nat Commun. 4:1953 (2013)).Recombinant relaxin hormone is in development for treatment of heartfailure. Analogs of relaxin, such as H2:A(4-24)(F23A) (see Chan et al.,J. Biol. Chem., 287:41152 (2012)), have also been found to have potency.However, due to the short half-life of relaxin, it requires intravenousadministration. Small molecule agonists of the relaxin receptor arebeing developed that are orally bioavailable (Xiao et al., supra). Thosemolecules can be identified using a high-throughput screening assaydeveloped for the RXFP1 gene product (Chen et al., J Biomol Screen.18:670 (2013)). Since RXFP1 has been reported to have lower expressionin ectopic endometriotic tissues (see Table 1) and a deletion in theRXFP1 gene is associated with EN (FIG. 2 ), recombinant relaxin, relaxinanalogs or RXFP1 small molecule agonists may have therapeutic value incertain EN patients. For example, Xiao et al. (supra) reported thatrelaxin, via its RXFP1 receptor, regulates cellular processes such asextracellular matrix remodeling, cell invasiveness, and proliferation,which are also known to be involved in the pathology of endometriosis.Those skilled in the art also appreciate that therapeutics developed totarget a specific gene (or its products) can be administered on thebasis of a patient testing positive for a particular genetic variantthat impacts the gene target or another gene in the same molecularpathway. In some embodiments, a genetic test is developed as a companiondiagnostic test for a specific therapeutic.

RNA Therapeutics

The nucleic acids and/or variants of the disclosure, or nucleic acidscomprising their complementary sequence, can be used as antisenseconstructs to control gene expression in cells, tissues or organs. Themethodology associated with antisense techniques is well known to theskilled artisan, and is described and reviewed in Antisense DrugTechnology: Principles, Strategies, and Applications, Crooke, MarcelDekker Inc., New York (2001) In general, antisense nucleic acids aredesigned to be complementary to a region of mRNA expressed by a gene, sothat the antisense molecule hybridizes to the mRNA, thus blockingtranslation of the mRNA into protein Several classes of antisenseoligonucleotide are known to those skilled in the art, includingcleavers and blockers. The former bind to target RNA sites, activateintracellular nucleases (e.g., Rnase H or Rnase L) that cleave thetarget RNA. Blockers bind to target RNA, inhibit protein translation bysteric hindrance of the ribosomes. Examples of blockers include nucleicacids, morpholino compounds, locked nucleic acids and methylphosphonates(Thompson, Drug Discovery Today, 7:912-917 (2002)) Antisenseoligonucleotides are useful directly as therapeutic agents, and are alsouseful for determining and validating gene function, for example by geneknock-out or gene knock-down experiments. Antisense technology isfurther described in Lavery et al., Curr. Opin. Drug Discov. Devel.,6:561-569 (2003), Stephens et al., Curr. Opin. Mol Ther., 5:118-122(2003), Kurreck, Eur. J. Biochem., 270:1628-44 (2003), Dias et al, Mol.Cancer Ter., 1:347-55 (2002), Chen, Methods Mol. Med., 75:621-636(2003), Wang et al., Curr. Cancer Drug Targets, 1:177-96 (2001), andBennett, Antisense Nucleic Acid Drug. Dev., 12 215-24 (2002).

The variants described herein can be used for the selection and designof antisense reagents that are specific for particular variants (e.g.,particular genetic variations or polymorphic markers in linkagedisequilibrium with particular genetic variations). Using informationabout the variants described herein, antisense oligonucleotides or otherantisense molecules that specifically target mRNA molecules that containone or more variants of the disclosure can be designed. In this manner,expression of mRNA molecules that contain one or more variants of thepresent disclosure (markers and/or haplotypes) can be inhibited orblocked. In some embodiments, the antisense molecules are designed tospecifically bind a particular allelic form (i.e., one or severalvariants (alleles and/or haplotypes)) of the target nucleic acid,thereby inhibiting translation of a product originating from thisspecific allele or haplotype, but which do not bind other or alternatevariants at the specific polymorphic sites of the target nucleic acidmolecule.

As antisense molecules can be used to inactivate mRNA so as to inhibitgene expression, and thus protein expression, the molecules can be usedto treat EN. The methodology can involve cleavage by means of ribozymescontaining nucleotide sequences complementary to one or more regions inthe mRNA that attenuate the ability of the mRNA to be translated. SuchmRNA regions include, for example, protein-coding regions, in particularprotein-coding regions corresponding to catalytic activity, substrateand/or ligand binding sites, or other functional domains of a protein.

The phenomenon of RNA interference (RNAi) has been actively studied forthe last decade, since its original discovery in C. elegans (Fire etal., Nature, 391:806-11 (1998)), and in recent years its potential usein treatment of human disease has been actively pursued (reviewed in Kim& Rossi, Nature Rev. Genet., 8:173-204 (2007)). RNA interference (RNAi),also called gene silencing, is based on using double-stranded RNAmolecules (dsRNA) to turn off specific genes. In the cell, cytoplasmicdouble-stranded RNA molecules (dsRNA) are processed by cellularcomplexes into small interfering RNA (siRNA). The siRNA guide thetargeting of a protein-RNA complex to specific sites on a target mRNA,leading to cleavage of the mRNA (Thompson, Drug Discovery Today,7:912-917 (2002)). The siRNA molecules are typically about 20, 21, 22 or23 nucleotides in length. Thus, one aspect of the disclosure relates toisolated nucleic acid sequences, and the use of those molecules for RNAinterference, for example as small interfering RNA molecules (siRNA). Insome embodiments, the isolated nucleic acid sequences can be 18-26nucleotides in length, or 19-25 nucleotides in length, or 20-24nucleotides in length, or 21, 22 or 23 nucleotides in length.

Another pathway for RNAi-mediated gene silencing originates inendogenously encoded primary microRNA (pn-miRNA) transcripts, which areprocessed in the cell to generate precursor miRNA (pre-miRNA). ThesemiRNA molecules are exported from the nucleus to the cytoplasm, wherethey undergo processing to generate mature miRNA molecules (miRNA),which direct translational inhibition by recognizing target sites in the3′ untranslated regions of mRNAs, and subsequent mRNA degradation byprocessing P-bodies (reviewed in Kim & Rossi, Nature Rev. Genet.,8:173-204 (2007)).

Clinical applications of RNAi include the incorporation of syntheticsiRNA duplexes, which are approximately 20-23 nucleotides in size, andmay have 3′ overlaps of 2 nucleotides. Knockdown of gene expression isestablished by sequence-specific design for the target mRNA. Severalcommercial sites for optimal design and synthesis of such molecules areknown to those skilled in the art.

Other applications provide longer siRNA molecules (typically 25-30nucleotides in length, such as about 27 nucleotides), as well as smallhairpin RNAs (shRNAs; typically about 29 nucleotides in length). Thelatter are naturally expressed, as described in Amarzguioui et al. (FEBSLett., 579:5974-81 (2005)). Chemically synthetic siRNAs and shRNAs aresubstrates for in vivo processing, and in some cases provide more potentgene-silencing than shorter designs (Kim et al., Nature Biotechnol.,23:222-226 (2005); Siola et al., Nature Biotechnol., 23:227-231 (2005)).In general siRNAs provide for transient silencing of gene expression,because their intracellular concentration is diluted by subsequent celldivisions. By contrast, expressed shRNAs mediate long-term, stableknockdown of target transcripts, for as long as transcription of theshRNA takes place (Marques et al., Nature Biotechnol., 23:559-565(2006), Brummelkamp et al., Science, 296:550-553 (2002)).

Since RNAi molecules, including siRNA, miRNA and shRNA, act in asequence-dependent manner, variants described herein can be used todesign RNAi reagents that recognize specific nucleic acids comprisingspecific genetic variations, alleles and/or haplotypes, while notrecognizing nucleic acid sequences not comprising the genetic variation,or comprising other alleles or haplotypes. These RNAi reagents can thusrecognize and destroy the target nucleic acid sequences. As withantisense reagents, RNAi reagents can be useful as therapeutic agents(i.e., for turning off disease-associated genes or disease-associatedgene variants), but can also be useful for characterizing and validatinggene function (e.g., by gene knock-out or gene knock-down experiments).

Delivery of RNAi can be performed by a range of methodologies known tothose skilled in the art. Methods utilizing non-viral delivery includecholesterol, stable nucleic acid-lipid particle (SNALP), heavy-chainantibody fragment (Fab), aptamers and nanoparticles Viral deliverymethods include use of lentivirus, adenovirus and adeno-associated virusThe siRNA molecules are in some embodiments chemically modified toincrease their stability. This can include modifications at the 2′position of the ribose, including 2′-O-methylpunnes and2′-fluoropyrimidmes, which provide resistance to RNase activity. Otherchemical modifications are possible and known to those skilled in theart.

The following references provide a further summary of RNAi, andpossibilities for targeting specific genes using RNAi: Kim & Rossi, Nat.Rev. Genet., 8:173 (2007), Chen & Rajewsky, Nat. Rev. Genet., 8:93(2007), Reynolds, et al., Nat. Biotechnol., 22:326 (2004), Chi et al.,Proc. Natl. Acad. Sa. USA, 100:6343 (2003), Vickers et al., J. Biol.Chem., 278:7108 (2003), Agami, Curr. Opin. Chem. Biol., 6:829 (2002),Lavery, et al., Curr. Opin. Drug Discov. Devel., 6:561 (2003), Shi,Trends Genet., 19:9 (2003), Shuey et al., Drug Discov. Today, 7:1040(2002), McManus et al., Nat. Rev. Genet., 3:737 (2002), Xia et al., Nat.Biotechnol., 20:1006 (2002), Plasterk et al., Curr. Opin Genet. Dev.,10:562 (2000), Bosher et al., Nat. Cell Biol., 2:E31 (2000), and Hunter,Curr. Biol., 9:R440 (1999).

A genetic defect leading to increased predisposition or risk fordevelopment of EN or a defect causing the disease, can be correctedpermanently by administering to a subject carrying the defect a nucleicacid fragment that incorporates a repair sequence that supplies thenormal/wild-type nucleotide(s) at the site of the genetic defect. Suchsite-specific repair sequence can encompass an RNA/DNA oligonucleotidethat operates to promote endogenous repair of a subject's genomic DNA.The administration of the repair sequence can be performed by anappropriate vehicle, such as a complex with polyethelamine, encapsulatedin anionic liposomes, a viral vector such as an adenovirus vector, orother pharmaceutical compositions suitable for promoting intracellularuptake of the administered nucleic acid The genetic defect can then beovercome, since the chimeric oligonucleotides induce the incorporationof the normal sequence into the genome of the subject, leading toexpression of the normal/wild-type gene product. The replacement ispropagated, thus rendering a permanent repair and alleviation of thesymptoms associated with the disease or condition.

Double stranded oligonucleotides are formed by the assembly of twodistinct oligonucleotide sequences where the oligonucleotide sequence ofone strand is complementary to the oligonucleotide sequence of thesecond strand; such double stranded oligonucleotides are generallyassembled from two separate oligonucleotides (e.g., siRNA), or from asingle molecule that folds on itself to form a double stranded structure(e.g., shRNA or short hairpin RNA). These double strandedoligonucleotides known in the art all have a common feature in that eachstrand of the duplex has a distinct nucleotide sequence, wherein onlyone nucleotide sequence region (guide sequence or the antisensesequence) has complementarity to a target nucleic acid sequence and theother strand (sense sequence) comprises nucleotide sequence that ishomologous to the target nucleic acid sequence.

Double stranded RNA induced gene silencing can occur on at least threedifferent levels: (i) transcription inactivation, which refers to RNAguided DNA or histone methylation; (ii) siRNA induced mRNA degradation;and (iii) mRNA induced transcriptional attenuation. It is generallyconsidered that the major mechanism of RNA induced silencing (RNAinterference, or RNAi) in mammalian cells is mRNA degradation. RNAinterference (RNAi) is a mechanism that inhibits gene expression at thestage of translation or by hindering the transcription of specificgenes. Specific RNAi pathway proteins are guided by the dsRNA to thetargeted messenger RNA (mRNA), where they “cleave” the target, breakingit down into smaller portions that can no longer be translated intoprotein. Initial attempts to use RNAi in mammalian cells focused on theuse of long strands of dsRNA. However, these attempts to induce RNAi metwith limited success, due in part to the induction of the interferonresponse, which results in a general, as opposed to a target-specific,inhibition of protein synthesis. Thus, long dsRNA is not a viable optionfor RNAi in mammalian systems. Another outcome is epigenetic changes toa gene—histone modification and DNA methylation—affecting the degree thegene is transcribed.

More recently it has been shown that when short (18-30 bp) RNA duplexesare introduced into mammalian cells in culture, sequence-specificinhibition of target mRNA can be realized without inducing an interferonresponse. Certain of these short dsRNAs, referred to as small inhibitoryRNAs (“siRNAs”), can act catalytically at sub-molar concentrations tocleave greater than 95% of the target mRNA in the cell. A description ofthe mechanisms for siRNA activity, as well as some of its applicationsare described in Provost et al., EMBO J., 21:5864 (2002); Tabara et al.,Cell, 109:861 (2002); Martinez et al., Cell, 110:563 (2002); Hutvagner &Zamore, Science, 297:2056 (2002).

From a mechanistic perspective, introduction of long double stranded RNAinto plants and invertebrate cells is broken down into siRNA by a TypeIII endonuclease known as Dicer. Sharp, RNA interference-2001, GenesDev., 15:485 (2001). Dicer, a ribonuclease-III-like enzyme, processesthe dsRNA into 19-23 base pair short interfering RNAs withcharacteristic two base 3′ overhangs. Bernstein, Caudy, Hammond, &Hannon, Nature, 409:363 (2001). The siRNAs are then incorporated into anRNA-induced silencing complex (RISC) where one or more helicases unwindthe siRNA duplex, enabling the complementary antisense strand to guidetarget recognition (Nykanen, Haley, & Zamore, Cell, 107:309 (2001)).Upon binding to the appropriate target mRNA, one or more endonucleaseswithin the RISC cleaves the target to induce silencing. Elbashir,Lendeckel, & Tuschl, Genes Dev., 15:188 (2001).

Generally, the antisense sequence is retained in the active RISC complexand guides the RISC to the target nucleotide sequence by means ofcomplementary base-pairing of the antisense sequence with the targetsequence for mediating sequence-specific RNA interference. It is knownin the art that in some cell culture systems, certain types ofunmodified siRNAs can exhibit “off target” effects. It is hypothesizedthat this off-target effect involves the participation of the sensesequence instead of the antisense sequence of the siRNA in the RISCcomplex (see for example, Schwarz et al., Cell, 115:199 (2003)). In thisinstance the sense sequence is believed to direct the RISC complex to asequence (off-target sequence) that is distinct from the intended targetsequence, resulting in the inhibition of the off-target sequence. Inthese double stranded nucleic acid sequences, each strand iscomplementary to a distinct target nucleic acid sequence. However, theoff-targets that are affected by these dsRNAs are not entirelypredictable and are non-specific.

The term “siRNA” refers to small inhibitory RNA duplexes that induce theRNA interference (RNAi) pathway. These molecules can vary in length(generally between 18-30 basepairs) and contain varying degrees ofcomplementarity to their target mRNA in the antisense strand. Some, butnot all, siRNA have unpaired overhanging bases on the 5′ or 3′ end ofthe sense strand and/or the antisense strand. The term “siRNA” includesduplexes of two separate strands, as well as single strands that canform hairpin structures comprising a duplex region. Small interferingRNA (siRNA), sometimes known as short interfering RNA or silencing RNA,are a class of 20-25 nucleotide-long double-stranded RNA molecules thatplay a variety of roles in biology.

While the two RNA strands do not need to be completely complementary,the strands should be sufficiently complementary to hybridize to form aduplex structure. In some instances, the complementary RNA strand can beless than 30 nucleotides, less than 25 nucleotides in length, about 19to 24 nucleotides in length, or 20-23 nucleotides in length, including22 nucleotides in length. The dsRNA of the present disclosure canfurther comprise at least one single-stranded nucleotide overhang. ThedsRNA of the present disclosure can further comprise a substituted orchemically modified nucleotide. As discussed in detail below, the dsRNAcan be synthesized by standard methods known in the art.

siRNA can be divided into five (5) groups including non-functional,semi-functional, functional, highly functional, and hyper-functionalbased on the level or degree of silencing that they induce in culturedcell lines. As used herein, these definitions are based on a set ofconditions where the siRNA is transfected into the cell line at aconcentration of 100 nM and the level of silencing is tested at a timeof roughly 24 hours after transfection, and not exceeding 72 hours aftertransfection. In this context, “non-functional siRNA” are defined asthose siRNA that induce less than 50% (<50%) target silencing.“Semi-functional siRNA” induce 50-79% target silencing. “FunctionalsiRNA” are molecules that induce 80-95% gene silencing.“Highly-functional siRNA” are molecules that induce greater than 95%gene silencing. “Hyperfunctional siRNA” are a special class ofmolecules. For purposes of this document, hyperfunctional siRNA aredefined as those molecules that: (1) induce greater than 95% silencingof a specific target when they are transfected at subnanomolarconcentrations (i.e., less than one nanomolar); and/or (2) inducefunctional (or better) levels of silencing for greater than 96 hours.These relative functionalities (though not intended to be absolutes) canbe used to compare siRNAs to a particular target for applications suchas functional genomics, target identification and therapeutics.

microRNAs (miRNA) are single-stranded RNA molecules of about 21-23nucleotides in length, which regulate gene expression. miRNAs areencoded by genes that are transcribed from DNA but not translated intoprotein (non-coding RNA); instead they are processed from primarytranscripts known as pri-miRNA to short stem-loop structures calledpre-miRNA and finally to functional miRNA. Mature miRNA molecules arepartially complementary to one or more messenger RNA (mRNA) molecules,and their main function is to downregulate gene expression.

Antibody-Based Therapeutics

The present disclosure embodies agents that modulate a peptide sequenceor RNA expressed from a gene associated with EN. The term biomarker, asused herein, can comprise a genetic variation of the present disclosureor a gene product, for example, RNA and polypeptides, of any one of thegenes listed in FIGS. 1-4 . Such modulating agents include, but are notlimited to, proteins, peptides, peptidomimetics, peptoids, or any otherforms of a molecule, which bind to, and alter the signaling or functionassociated with the EN associated biomarker, have an inhibitory orstimulatory effect on the EN associated biomarkers, or have astimulatory or inhibitory effect on the expression or activity of the ENassociated biomarkers' ligands, for example, polyclonal antibodiesand/or monoclonal antibodies that specifically bind one form of the geneproduct but not to the other form of the gene product are also provided,or which bind a portion of either the variant or the reference geneproduct that contains the polymorphic site or sites.

In some embodiments, the present disclosure provides antibody-basedagents targeting EN associated biomarkers. The antibody-based agents inany suitable form of an antibody, e.g., monoclonal, polyclonal, orsynthetic, can be utilized in the therapeutic methods disclosed herein.The antibody-based agents include any target-binding fragment of anantibody and also peptibodies, which are engineered therapeuticmolecules that can bind to human drug targets and contain peptideslinked to the constant domains of antibodies. In some embodiments, theantibodies used for targeting EN associated biomarkers are humanizedantibodies. Methods for humanizing antibodies are well known in the art.In some embodiments, the therapeutic antibodies comprise an antibodygenerated against EN associated biomarkers described in the presentdisclosure, wherein the antibodies are conjugated to another agent oragents, for example, a cytotoxic agent or agents.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain antigen-binding sites that specifically bind anantigen. A molecule that specifically binds to a polypeptide of thedisclosure is a molecule that binds to that polypeptide or a fragmentthereof, but does not substantially bind other molecules in a sample,e.g., a biological sample, which naturally contains the polypeptide.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab′)2 fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The disclosure providespolyclonal and monoclonal antibodies that bind to a polypeptide of thedisclosure. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of a polypeptide ofthe disclosure. A monoclonal antibody composition thus typicallydisplays a single binding affinity for a particular polypeptide of thedisclosure with which it immunoreacts.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a desired immunogen, e.g., polypeptide of thedisclosure or a fragment thereof. The antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilizedpolypeptide. If desired, the antibody molecules directed against thepolypeptide can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein, Nature,256:495 (1975), the human B cell hybridoma technique (Kozbor et al.,Immunol. Today, 4:72 (1983)), the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985) Inc., pp.77-96) or trioma techniques. The technology for producing hybridomas iswell known (see generally Current Protocols in Immunology (1994) Coliganet al., (eds.) John Wiley & Sons, Inc., New York, N.Y.). Briefly, animmortal cell line (typically a myeloma) is fused to lymphocytes(typically splenocytes) from a mammal immunized with an immunogen asdescribed above, and the culture supernatants of the resulting hybridomacells are screened to identify a hybridoma producing a monoclonalantibody that binds a polypeptide of the disclosure.

Any of the many well-known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody to a polypeptide of the disclosure (see, e.g.,Current Protocols in Immunology, supra; Galfre et al., Nature, 266:55052(1977); R. H. Kenneth, in Monoclonal Antibodies: A New Dimension InBiological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); andLerner, J. Biol. Med., 54:387 (1981)). Moreover, the ordinarily skilledworker can appreciate that there are many variations of such methodsthat also would be useful. Alternative to preparing monoclonalantibody-secreting hybridomas, a monoclonal antibody to a polypeptide ofthe disclosure can be identified and isolated by screening a recombinantcombinatorial immunoglobulin library (e.g., an antibody phage displaylibrary) with the polypeptide to thereby isolate immunoglobulin librarymembers that bind the polypeptide. Kits for generating and screeningphage display libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP^(a) Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication Nos. WO92/18619, WO 91/17271, WO 92/20791, WO 92/15679; WO 93/01288, WO92/01047, WO 92/09690, and WO 90/02809; Fuchs et al., Bio/Technology,9:1370 (1991); Hay et al., Hum. Antibod. Hybndomas, 3:81 (1992); Huse etal., Science, 246:1275 (1989); and Griffiths et al., EMBO J., 12:725(1993).

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the disclosure. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart.

In general, antibodies of the disclosure (e.g., a monoclonal antibody)can be used to isolate a polypeptide of the disclosure by standardtechniques, such as affinity chromatography or immunoprecipitation. Apolypeptide-specific antibody can facilitate the purification of naturalpolypeptide from cells and of recombinants produced polypeptideexpressed in host cells Moreover, an antibody specific for a polypeptideof the disclosure can be used to detect the polypeptide (e.g., in acellular lysate, cell supernatant, or tissue sample) in order toevaluate the abundance and pattern of expression of the polypeptide.Antibodies can be used diagnostically, prognostically, ortheranostically to monitor protein levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. The antibody can be coupled to adetectable substance to facilitate its detection. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotnazinylamine fluorescein, dansylchloride or phycoerythnn; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H. Antibodies can also be useful inpharmacogenomic analysis. In such embodiments, antibodies againstvariant proteins encoded by nucleic acids according to the disclosure,such as variant proteins that are encoded by nucleic acids that containat least one genetic variation of the disclosure, can be used toidentify individuals that can benefit from modified treatmentmodalities.

Antibodies can furthermore be useful for assessing expression of variantproteins in disease states, such as in active stages of a disease, or inan individual with a predisposition to a disease related to the functionof the protein, in particular EN. Antibodies specific for a variantprotein of the present disclosure that is encoded by a nucleic acid thatcomprises at least one polymorphic marker or haplotype as describedherein can be used to screen for the presence of the variant protein,for example to screen for a predisposition to EN as indicated by thepresence of the variant protein.

Antibodies can be used in other methods. Thus, antibodies are useful asscreening tools for evaluating proteins, such as variant proteins of thedisclosure, in conjunction with analysis by electrophoretic mobility,isoelectric point, tryptic or other protease digest, or for use in otherphysical assays known to those skilled in the art. Antibodies can alsobe used in tissue typing. In one such embodiment, a specific variantprotein has been correlated with expression in a specific tissue type,and antibodies specific for the variant protein can then be used toidentify the specific tissue type.

Subcellular localization of proteins, including variant proteins, canalso be determined using antibodies, and can be applied to assessaberrant subcellular localization of the protein in cells in varioustissues. Such use can be applied in genetic testing, but also inmonitoring a particular treatment modality. In the case where treatmentis aimed at correcting the expression level or presence of the variantprotein or aberrant tissue distribution or, for instance, endometrial orblood cell expression of the variant protein, antibodies specific forthe variant protein or fragments thereof can be used to monitortherapeutic efficacy.

Antibodies are further useful for inhibiting variant protein function,for example by blocking the binding of a variant protein to a bindingmolecule or partner. Such uses can also be applied in a therapeuticcontext in which treatment involves inhibiting a variant protein'sfunction. An antibody can be for example be used to block orcompetitively inhibit binding, thereby modulating (i.e., agonizing orantagonizing) the activity of the protein. Antibodies can be preparedagainst specific protein fragments containing sites for specificfunction or against an intact protein that is associated with a cell orcell membrane.

The present disclosure also embodies the use of any pharmacologic agentthat can be conjugated to an antibody or an antibody binding fragment,and delivered in active form. Examples of such agents includecytotoxins, radioisotopes, hormones such as a steroid, anti-metabolitessuch as cytosines, and chemotherapeutic agents. Other embodiments caninclude agents such as a coagulant, a cytokine, growth factor, bacterialendotoxin or a moiety of bacterial endotoxin. The targetingantibody-based agent directs the toxin to, and thereby selectivelymodulates the cell expressing the targeted surface receptor. In someembodiments, therapeutic antibodies employ cross-linkers that providehigh in vivo stability (Thorpe et al., Cancer Res., 48:6396 (1988)). Inany event, it is proposed that agents such as these can, if desired, besuccessfully conjugated to antibodies or antibody binding fragments, ina manner that can allow their targeting, internalization, release orpresentation at the site of the targeted cells expressing the ENassociated biomarkers using known conjugation technology. Foradministration in vivo, for example, an antibody can be linked with anadditional therapeutic payload, such as radionuclide, an enzyme, animmunogenic epitope, or a cytotoxic agent, including bacterial toxins(diphtheria or plant toxins, such as ricin). The in vivo half-life of anantibody or a fragment thereof can be increased by pegylation throughconjugation to polyethylene glycol.

Methods of Treatment

One embodiment of the present disclosure relates to methods of usingpharmaceutical compositions and kits comprising agents that can reduceor increase the function and/or activity of polypeptides and/or nucleicacids that are associated with EN to inhibit or decrease EN progression.Another embodiment of the present disclosure provides methods,pharmaceutical compositions, and kits for the treatment of animalsubjects. The term “animal subject” as used herein includes humans aswell as other mammals. The term “treating” as used herein includesachieving a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying cause of EN. Also, a therapeutic benefit is achieved with theeradication or amelioration of one or more of the physiological symptomsassociated EN such that an improvement is observed in the animalsubject, notwithstanding the fact that the animal subject can still beafflicted with EN.

For embodiments where a prophylactic benefit is desired, apharmaceutical composition of the disclosure can be administered to asubject at risk of developing EN, or to a subject reporting one or moreof the physiological symptoms of EN, even though a screening of thecondition cannot have been made. Administration can prevent EN fromdeveloping, or it can reduce, lessen, shorten and/or otherwiseameliorate the progression of EN, or symptoms that develop. Thepharmaceutical composition can modulate a target EN associatedbiomarker. Wherein, the term modulate includes inhibition of ENassociated biomarkers or alternatively activation of EN associatedbiomarkers.

Reducing the activity and/or function of polypeptides and/or nucleicacids found to be associated with EN, is also referred to as“inhibiting” the polypeptides and/or nucleic acids. The term “inhibits”and its grammatical conjugations, such as “inhibitory,” do not requirecomplete inhibition, but refer to a reduction in EN associatedbiomarkers' activities. In some embodiments, such reduction is by atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 75%, at least 90%, and can be by at least 95% of theactivity of the enzyme in the absence of the inhibitory effect, e.g., inthe absence of an inhibitor. Conversely, the phrase “does not inhibit”and its grammatical conjugations refer to situations where there is lessthan 20%, less than 10%, and can be less than 5%, of reduction in enzymeactivity in the presence of the agent. Further the phrase “does notsubstantially inhibit” and its grammatical conjugations refer tosituations where there is less than 30%, less than 20%, and in someembodiments less than 10% of reduction in enzyme activity in thepresence of the agent.

Increasing the activity and/or function of polypeptides and/or nucleicacids found to be associated with EN, is also referred to as“activating” the polypeptides and/or nucleic acids. The term “activated”and its grammatical conjugations, such as “activating,” do not requirecomplete activation, but refer to an increase in EN associatedbiomarkers' activities. In some embodiments such increase is by at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, and can be by at least 95% of the activity of theenzyme in the absence of the activation effect, e.g., in the absence ofan activator. Conversely, the phrase “does not activate” and itsgrammatical conjugations refer to situations where there can be lessthan 20%, less than 10%, and less than 5%, of an increase in enzymeactivity in the presence of the agent. Further the phrase “does notsubstantially activate” and its grammatical conjugations refer tosituations where there is less than 30%, less than 20%, and in someembodiments less than 10% of an increase in enzyme activity in thepresence of the agent.

The ability to reduce enzyme activity is a measure of the potency or theactivity of an agent, or combination of agents, towards or against theenzyme. Potency can be measured by cell free, whole cell and/or in vivoassays in terms of IC50, Ki and/or ED50 values. An IC50 value representsthe concentration of an agent to inhibit enzyme activity by half (50%)under a given set of conditions. A Ki value represents the equilibriumaffinity constant for the binding of an inhibiting agent to the enzyme.An ED50 value represents the dose of an agent to affect a half-maximalresponse in a biological assay. Further details of these measures willbe appreciated by those of ordinary skill in the art, and can be foundin standard texts on biochemistry, enzymology, and the like.

The present disclosure also includes kits that can be used to treat EN.These kits comprise an agent or combination of agents that inhibits anEN associated biomarker and in some embodiments instructions teachingthe use of the kit according to the various methods and approachesdescribed herein. Such kits can also include information, such asscientific literature references, package insert materials, clinicaltrial results, and/or summaries of these and the like, which indicate orestablish the activities and/or advantages of the agent. Suchinformation can be based on the results of various studies, for example,studies using experimental animals involving in vivo models and studiesbased on human clinical trials. Kits described herein can be provided,marketed and/or promoted to health providers, including physicians,nurses, pharmacists, formulary officials, and the like.

Kits

Kits useful in the methods of the disclosure comprise components usefulin any of the methods described herein, including for example, primersfor nucleic acid amplification, hybridization probes for detectinggenetic variation, or other marker detection, restriction enzymes,nucleic acid probes, optionally labeled with suitable labels,allele-specific oligonucleotides, antibodies that bind to an alteredpolypeptide encoded by a nucleic acid of the disclosure as describedherein or to a wild type polypeptide encoded by a nucleic acid of thedisclosure as described herein, means for amplification of geneticvariations or fragments thereof, means for analyzing the nucleic acidsequence of nucleic acids comprising genetic variations as describedherein, means for analyzing the amino acid sequence of a polypeptideencoded by a genetic variation, or a nucleic acid associated with agenetic variation, etc. The kits can for example include necessarybuffers, nucleic acid primers for amplifying nucleic acids, and reagentsfor allele-specific detection of the fragments amplified using suchprimers and necessary enzymes (e.g., DNA polymerase). Additionally, kitscan provide reagents for assays to be used in combination with themethods of the present disclosure, for example reagents for use withother screening assays for EN.

In some embodiments, the disclosure pertains to a kit for assaying asample from a subject to detect the presence of a genetic variation,wherein the kit comprises reagents necessary for selectively detectingat least one particular genetic variation in the genome of theindividual. In some embodiments, the disclosure pertains to a kit forassaying a sample from a subject to detect the presence of at leastparticular allele of at least one polymorphism associated with a geneticvariation in the genome of the subject. In some embodiments, thereagents comprise at least one contiguous oligonucleotide thathybridizes to a fragment of the genome of the individual comprising atleast genetic variation. In some embodiments, the reagents comprise atleast one pair of oligonucleotides that hybridize to opposite strands ofa genomic segment obtained from a subject, wherein each oligonucleotideprimer pair is designed to selectively amplify a fragment of the genomeof the individual that includes at least one genetic variation, or afragment of a genetic variation. Such oligonucleotides or nucleic acidscan be designed using the methods described herein. In some embodiments,the kit comprises one or more labeled nucleic acids capable ofallele-specific detection of one or more specific polymorphic markers orhaplotypes with a genetic variation, and reagents for detection of thelabel. In some embodiments, a kit for detecting SNP markers can comprisea detection oligonucleotide probe, that hybridizes to a segment oftemplate DNA containing a SNP polymorphisms to be detected, an enhanceroligonucleotide probe, detection probe, primer and/or an endonuclease,for example as described by Kutyavin et al. (Nucleic Acid Res., 34:e128(2006)).

In some embodiments, the DNA template is amplified by any means of thepresent disclosure, prior to assessment for the presence of specificgenetic variations as described herein. Standard methods well known tothe skilled person for performing these methods can be utilized, and arewithin scope of the disclosure. In one such embodiment, reagents forperforming these methods can be included in the reagent kit.

In a further aspect of the present disclosure, a pharmaceutical pack(kit) is provided, the pack comprising a therapeutic agent and a set ofinstructions for administration of the therapeutic agent to humansscreened for one or more variants of the present disclosure, asdisclosed herein. The therapeutic agent can be a small molecule drug, anantibody, a peptide, an antisense or RNAi molecule, or other therapeuticmolecules as described herein. In some embodiments, an individualidentified as a carrier of at least one variant of the presentdisclosure is instructed to take a prescribed dose of the therapeuticagent. In one such embodiment, an individual identified as a carrier ofat least one variant of the present disclosure is instructed to take aprescribed dose of the therapeutic agent. In some embodiments, anindividual identified as a non-carrier of at least one variant of thepresent disclosure is instructed to take a prescribed dose of thetherapeutic agent.

Also provided herein are articles of manufacture, comprising a probethat hybridizes with a region of human chromosome as described hereinand can be used to detect a polymorphism described herein. For example,any of the probes for detecting polymorphisms described herein can becombined with packaging material to generate articles of manufacture orkits. The kit can include one or more other elements including:instructions for use; and other reagents such as a label or an agentuseful for attaching a label to the probe. Instructions for use caninclude instructions for screening applications of the probe for makinga diagnosis, prognosis, or theranosis to EN in a method describedherein. Other instructions can include instructions for attaching alabel to the probe, instructions for performing in situ analysis withthe probe, and/or instructions for obtaining a sample to be analyzedfrom a subject. In some cases, the kit can include a labeled probe thathybridizes to a region of human chromosome as described herein.

The kit can also include one or more additional reference or controlprobes that hybridize to the same chromosome or another chromosome orportion thereof that can have an abnormality associated with aparticular endophenotype. A kit that includes additional probes canfurther include labels, e.g., one or more of the same or differentlabels for the probes. In other embodiments, the additional probe orprobes provided with the kit can be a labeled probe or probes. When thekit further includes one or more additional probe or probes, the kit canfurther provide instructions for the use of the additional probe orprobes. Kits for use in self-testing can also be provided. Such testkits can include devices and instructions that a subject can use toobtain a biological sample (e.g., buccal cells, blood) without the aidof a health care provider. For example, buccal cells can be obtainedusing a buccal swab or brush, or using mouthwash.

Kits as provided herein can also include a mailer (e.g., a postage paidenvelope or mailing pack) that can be used to return the sample foranalysis, e.g., to a laboratory. The kit can include one or morecontainers for the sample, or the sample can be in a standard bloodcollection vial. The kit can also include one or more of an informedconsent form, a test requisition form, and instructions on how to usethe kit in a method described herein. Methods for using such kits arealso included herein. One or more of the forms (e.g., the testrequisition form) and the container holding the sample can be coded, forexample, with a bar code for identifying the subject who provided thesample.

In some embodiments, an in vitro screening test can comprise one or moredevices, tools, and equipment configured to collect a genetic samplefrom an individual. In some embodiments of an in vitro screening test,tools to collect a genetic sample can include one or more of a swab, ascalpel, a syringe, a scraper, a container, and other devices andreagents designed to facilitate the collection, storage, and transportof a genetic sample. In some embodiments, an in vitro screening test caninclude reagents or solutions for collecting, stabilizing, storing, andprocessing a genetic sample.

Such reagents and solutions for nucleotide collecting, stabilizing,storing, and processing are well known by those of skill in the art andcan be indicated by specific methods used by an in vitro screening testas described herein. In some embodiments, an in vitro screening test asdisclosed herein, can comprise a microarray apparatus and reagents, aflow cell apparatus and reagents, a multiplex nucleotide sequencer andreagents, and additional hardware and software necessary to assay agenetic sample for certain genetic markers and to detect and visualizecertain genetic markers.

The present disclosure further relates to kits for using antibodies inthe methods described herein. This includes, but is not limited to, kitsfor detecting the presence of a variant protein in a test sample. Oneembodiment comprises antibodies such as a labeled or labelable antibodyand a compound or agent for detecting variant proteins in a biologicalsample, means for determining the amount or the presence and/or absenceof variant protein in the sample, and means for comparing the amount ofvariant protein in the sample with a standard, as well as instructionsfor use of the kit. In certain embodiments, the kit further comprises aset of instructions for using the reagents comprising the kit.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The followingreferences contain embodiments of the methods and compositions that canbe used herein: The Merck Manual of Diagnosis and Therapy, 18th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2);Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007(ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Standard procedures of the present disclosure are described, e.g., inManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrooket al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis etal., Basic Methods in Molecular Biology, Elsevier Science Publishing,Inc., New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques, Vol. 152, S. L. Berger and A. R. Kimmerl (eds.),Academic Press Inc., San Diego, USA (1987)). Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols inImmunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manualof Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), and Animal Cell Culture Methods (Methods in CellBiology, Vol. 57, Jennie P. Mather and David Barnes editors, AcademicPress, 1st edition, 1998), which are all incorporated by referenceherein in their entireties.

It should be understood that the following examples should not beconstrued as being limiting to the particular methodology, protocols,and compositions, etc., described herein and, as such, can vary. Thefollowing terms used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of theembodiments disclosed herein.

Disclosed herein are molecules, materials, compositions, and componentsthat can be used for, can be used in conjunction with, can be used inpreparation for, or are products of methods and compositions disclosedherein. It is understood that when combinations, subsets, interactions,groups, etc. of these materials are disclosed and while specificreference of each various individual and collective combinations andpermutation of these molecules and compounds cannot be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a nucleotide or nucleic acid is disclosed and discussed anda number of modifications that can be made to a number of moleculesincluding the nucleotide or nucleic acid are discussed, each and everycombination and permutation of nucleotide or nucleic acid and themodifications that are possible are specifically contemplated unlessspecifically indicated to the contrary. This concept applies to allaspects of this application including, but not limited to, steps inmethods of making and using the disclosed molecules and compositions.Thus, if there are a variety of additional steps that can be performedit is understood that each of these additional steps can be performedwith any specific embodiment or combination of embodiments of thedisclosed methods, and that each such combination is specificallycontemplated and should be considered disclosed.

Those skilled in the art can recognize, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

It is understood that the disclosed methods and compositions are notlimited to the particular methodology, protocols, and reagents describedas these can vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present disclosure which canbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the meanings that would be commonly understood by one of skill inthe art in the context of the present specification.

It should be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “anucleotide” includes a plurality of such nucleotides; reference to “thenucleotide” is a reference to one or more nucleotides and equivalentsthereof known to those skilled in the art, and so forth.

The term “and/or” shall in the present context be understood to indicatethat either or both of the items connected by it are involved. Whilepreferred embodiments of the present disclosure have been shown anddescribed herein, it can be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions can now occur to those skilled inthe art without departing from the disclosure. It should be understoodthat various alternatives to the embodiments of the disclosure describedherein can be employed in practicing the disclosure. It is intended thatthe following claims define the scope of the disclosure and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The invention will be further described by the following non-limitingexamples.

Example 1

The present invention is not based on finding common variants thatincrease the risk for or cause disease, but rather rare variants. Inthis context ‘rare’ refers to variants that are present/observed indisease cohorts at a certain frequency but never or almost never innormal (non-disease or unaffected) subjects. ‘Rare’ refers to thefrequency in normal cohorts, but not necessarily in disease cohorts.Thus, a variant may actually be ‘common’ in the disease cohort but ifabsent or present at low frequency in a normal cohort, it would beclassed as a ‘rare’ variant. The present invention is based on adiscovery method that differs from traditional ‘common variant’ studieswhich, by design, typically identify weak associations between variantsand disease (e.g., with low odds ratios of <1.5), since the variantsbeing studied are known, in advance, to be present at an appreciablefrequency in normal/unaffected subjects. As an example, consider theTGFBR3 deletion found in 3/100 EN cases but in 0/1,005 normal subjects(see FIG. 5 ). This deletion is clearly ‘very rare’ in normal subjects(found in the study described herein at 0% frequency) but this is not soin the EN cohort (the deletion is found at a frequency of 3%). Whilethere is no specific frequency cutoff that defines a variant as rare orcommon, it is generally accepted by those skilled in the art and thegenetics field that rare variants occur at <1% frequency in the generalpopulation (i.e., the presence of the TGFBR3 deletion in the EN cohortat 3% frequency would be considered a ‘common’ variant by this generalcutoff used for the population at large). The discovery methodologydescribed herein is able to detect rare variants because it is notlimited to the analysis of previously known variants, as is the casewith the common variant SNP-based genome-wide association studies (GWAS)that have been employed by many skilled in the art for the discovery ofgenes that are causal or associated with the disease being studied.Thus, for a complex disease such EN, the present method identifiessingle highly causal genes and rare gene variations including intragenicregions or nongenic (intergenic) regions. Data was generated on thebasis of a comparison of copy number variants (CNVs) identified in 2cohorts: 1,005 Normal individuals (Normal Variation Engine—NVE); and 100endometriosis (EN) cases.

Genomic DNA Sample Hybridization

Genomic DNA samples from individuals within the Normal cohort (NVE‘test’ subjects) and from the Endo cohort (Endo ‘test’ subjects) werehybridized against a single, sex-matched reference individual asfollows. Reference DNA samples were labeled with Cy5 and test subjectDNA samples were labeled with Cy3. After labeling, samples were combinedand co-hybridized to Agilent 1M feature oligonucleotide microarrays,design ID 021529 (Agilent Product Number G4447A) using standardconditions (array Comparative Genomic Hybridization-aCGH).Post-hybridization, arrays were scanned at 2 μm resolution, usingAgilent's DNA microarray scanner, generating tiff images for lateranalysis.

All tiff images were analyzed using Agilent Feature Extraction (FE)software, with the following settings:

Human Genome Freeze: hg18:NCBI36:Mar2006FE version: 10.7.3.1Grid/design file: 021529_D_F_20091001

Protocol: CGH_107_Sep09

This procedure generates a variety of output files, containing about1,000,000 rows of data, each corresponding to a specific feature on thearray. This file was used to perform CNV calling using DNAcopy, an opensource software package implemented in R via BioConductor(http://www.bioconductor.org/packages/release/bioc/html/DNAcopy.html).Losses or gains were determined according to a threshold log₂ ratio,which was set at −/+0.35. In other words, all losses with a log₂ ratiovalue <=−0.35 were counted, as were all gains with a log₂ ratio >=+0.35.All log₂ ratio values were determined according to Cy3/Cy5(Test/Reference). A minimum probe threshold for CNV-calling was set at 2(2 consecutive probes were sufficient to call a CNV). A CNV list wasthus generated for each individual in the 2 cohorts.

There were a total of 162,316 CNVs in the NVE cohort of 1,005individuals (an average of 162 CNVs per individual). These CNVs (many ofwhich appeared in multiple individuals) were ‘merged’ into a master list(NVE-master) of non-redundant CNV subregions, according to the presenceor absence of the CNV subregion in individuals within the cohort. Usingthis approach, the NVE-master list has 14,693 distinct CNV subregions,some of which are uniquely present in a single individual and some ofwhich are present in multiple individuals. For example, consider 3individuals within the NVE cohort with the following hypothetical CNVs:

-   -   A. Chr1:1-100,000;    -   B. Chr1:10,001-100,000;    -   C. Chr1:1-89,999;

In the master list, these would be merged into 3 distinct CNVsubregions, as follows:

CNV subregion 1 Chr1: 1-10,000 Patients A, C CNV subregion 2 Chr1:10,001-89,999 Patients A, B, C CNV subregion 3 Chr1: 90,000-100,000Patients A, B

There were a total of 16,577 CNVs in the EN cohort of 100 individuals(an average of 166 CNVs per individual). These CNVs (many of whichappeared in multiple individuals) were ‘merged’ into a master list(EN-master) of non-redundant CNV subregions, according to the presenceor absence of the CNV subregion in individuals within the cohort. Usingthis approach, the EN-master list has 3,408 distinct CNV subregions,some of which are uniquely present in a single individual and some ofwhich are present in multiple individuals.

CNV subregions of interest were obtained after:

-   -   1. Annotation in order to attach to each CNV region relevant        information regarding overlap with known genes and exons;    -   2. A calculation of the odds ratio (OR) for each CNV subregion,        according to the following formula:

*OR=(EN/(100−EN))/(NVE/(1005−NVE))

-   -   where:    -   EN=number of EN individuals with CNV subregion of interest    -   NVE=number of NVE individuals with CNV subregion of interest.

As an illustrative example, consider the CNV subregionchr1:92013987-92020793, which is found in 0 individuals in the NVEcohort and 3 individuals in the EN cohort.

The OR is:

(3.5/(97.5))/(0.5/(1005.5))=72.19

Note that, by one convention, if either of NVE or EN=0, a value of 0.5is added to all 4 entries in the main formula above, in order to avoiddealing with infinities. This has the effect of artificially lowering ORvalues in cases where no individuals within the NVE have the CNV. Thismethod is also used when calculating the Fisher's 2-tailed Exact Test(FET) in the event that any one of the variables is zero.

CNV subregions/genes that fulfill one of the following criteria wereidentified (see FIG. 1 ):

-   -   1. Strong biology linking the CNV subregion and/or the gene it        overlaps, with known pathways/mechanisms or biology in EN (in        some cases, statistical evidence is lacking but does not exclude        the CNV subregion as a candidate);    -   2. Statistical analysis without obvious biological connection        (FDR adjusted p-value <=0.05);    -   3. A combination of statistical significance and biology.

It can be appreciated by those skilled in the art that the number of ENcandidate CNV subregions, irrespective of category, may increase ordecrease as additional EN cohorts are analyzed.

FIG. 1 lists exemplary CNVs of interest. FIG. 2 shows the CNVcoordinates for the actual CNV subregions found to be unique orsignificantly different between the disease and normal cohorts, asopposed to FIG. 1 , which lists the original CNV coordinates. FIG. 2also details whether genic CNV subregions of interest overlap an exon ornot and the number of normal subjects and the number of disease subjectsthat harbor the relevant CNV subregion, reports Fisher's 2-tailed ExactTest (FET) and odds ratio (OR), and provides the category under whichthe CNV subregion falls with respect to statistical and/or biologicalsignificance as discussed above. FIG. 3 represents a non-redundant listfor all genes listed in FIG. 2 . For all genes listed in FIG. 2 , FIG. 4represents a non-redundant list of relevant RNA transcripts. Table Isummarizes biology and uterus expression data for genes impacted byCNVs/CNV subregions of interest (NA=data or information ‘notavailable’).

The sequence file 3886.001PRV ST25.txt contains genomic sequenceinformation for (in the following order): All distinct CNVs listed inFIG. 1 (SEQ ID NOs: 1-47) and the full genomic extent of the transcriptslisted in FIG. 4 (SEQ ID NOs: 48-149).

Examples of Sequences:

Sequence entry starts:SEQ_ID 1 = 6,806 bp CNV (loss) involving an intron of the gene TGFBR3:<210> 1 <211> 6807 <212> DNA <213> Homo sapiens <400> 1actgaaacat tcattttcca agattccttt ccaaattcaa ctccatggtc tttcttttgt 60ctgttttgct aaagggcaaa atactaacct gtttccaagc tattcagcaa tattttggca 120caggtaatcc agtaagagaa ttgtttgtat agaacagagc tgatgttaat aacagggtgc 180........................................................(sequencetruncated for brevity)aaagcactgc ttgcacatcg actgacctga ggttgagagg aggagcagag gaatgtggaa 6720aggattgttt ttaccttcat taagtgttaa atttacctaa gactcccaga gagtcaatgc 6780tcttcaggaa ctattctgag gcagaaa 6807 Sequence entry ends.Sequence entry starts:SEQ_ID 149 = GPR111, transcript NM_153839, which is 41,208 bp in length:<210> 149 <211> 41208 <212> DNA <213> Homo sapiens <400> 149gcaaggtatg acagaagaga gccagtaatg cccatatctc cagctgccag ggagtggggc 60aaatttgccg gtgagtttgt ctttggccag ccacagtgag tagtcccagg tcctccttct 120ccttccagtg acttcctttg gagaggaatt tcagagatga gcagccactc atgattttgt 180.................................(sequence truncated for brevity)ccttttggtc tttttgtgtg catatgtata tgttttgggg aatggggtat tcacttttgt 41100tactcactgt gttactcact tttgtatgcc catagtgcag agcatggtgc cttgtacata 41160gagtatgttc ggtaaatatg tgcaataaaa agtcctttga ttacacaa 41208Sequence entry ends.

TABLE I Biological notes for genes impacted by CNVs/CNV Subregions ofInterest CHR START STOP Gene_ symbol Expression_data_uterusBiology_notes PubMed_PMID_ No 1 92,013,987 92,020,793 TGFBR3 yes One ofseveral TGF beta superfamily 11912285, 15745937, members involved inendometrial 16613890, 16621788, function, links to CNV-identified16885531, 21261473, endometriosis candidates TGFB1I1 22562249, 23242524(Hic-5) and PTK2 (FAK) 23 149,901,706 149,902,701 HMGB3 yes Familymember HMGB1 expressed 18483013, 22014880 in endometrium and regulatedby estradiol, progesterone, and nitric oxide 23 149,902,702 149,904,265HMGB3 yes Family member HMGB1 expressed 18483013, 22014880 inendometrium and regulated by estradiol, progesterone, and nitic oxide 696,610,680 96,625,609 FUT9 yes Link between FUT9 7627975, 23192350(fucosyltransferase) and SELE (e-selectin) and endometrial, 16 PubMedcitations for “fucosyltransferase AND endometrial”, 15 PubMed citationsfor “e-selectin AND endometrial”, 7 PubMed citations “e-selectin ANDendometriosis” 7 31,844,434 31,851,158 PDE1C no Regulates collagenhomeostasis and 9366577, 18335582, is a key regulator of pathological21112686, 21148428, vascular remodeling and CNV- 21576276, 21962439,identified endometriosis candidate 22472216 TGFB1I1 (Hic-5) is linked tovascular remodeling and endometriosis, PDE1C maps to linkage peak7p13-15 for endometriosis 10 59,620,764 59,630,493 IPMK no IPMK linkedto p53 and 95 PubMed 21284988, 23550211, citations for “p53 AND 23708509endometriosis”, IPMK linked to mTor and 8 PubMed citations for “mtor ANDendometriosis” plus 111 PubMed citations for “mtor AND endometrial” 2233,285,559 233,298,235 GIGYF2 no IGF-1 expression linked to 12771153,20844834, endometriosis and GYGYF1 and 23776368 GYGYF2 are transientlylinked to IGF-I receptors by the Grb10 adapter protein (GRB10) followingIGF-I stimulation, GYGYF2 has also been incorrectly identified as aParkinson's disease gene (PARK11) 6 138,516,944 138,520,155 intergenicno Intergenic and located near 19496786 KIAA1244 (BIG3), which is linkedto estrogen/ER signaling in breast cancer 11 44,056,561 44,058,123 ACCSyes NA 11470512, 22543105 7 153,645,525 153,647,352 DPP6 no NA NA 6120,674,750 120,680,729 intergenic NA NA NA 16 31,356,038 31,434,641TGFB1I1 yes TGFB1I1 (Hic-5) is a coactivator of 19389829, 21715447, theprogesterone receptor and may be 22472216, 22529104 involved inprogesterone resistance observed in some endometriosis patients, linkedto vascular remodeling, interacts with CNV- identified endometriosiscandidate PTK2 and acts as a scaffold for focal adhesions, TP53 andCDKN1A (p21, WAF1, CIP1) genotypes correlate with endometriosis andTGFB1I1 transactivates CDKN1A, and potential pharmacolgoical link toHDAC inhibitors, links to CNV- identified endometriosis candidatesTGFBR3 (betaglycan) PDE1C, and PTK2 (FAK) 8 142,060,703 142,065,735 PTK2no PTK2 (FAK) is implicated in 17543958, 17550607, endometriosis andendometrial 18294638, 19471549, cancer, links to CNV-identified20869705, 21058027, endometriosis candidates TGFB1I1 21900245, 23242524(Hic-5) and TGFBR3 (betaglycan) 4 129,189,476 129,451,283 PGRMC2 yesGain of several genes, PGRMC2 has 22307145, 22355044, links toendometriosis and 23276631, 23522067 progesterone, and expression levelscorrelate with diminished ovarian reserve (see also CNV-identifiedendometriosis candidate MYADML) 19 41,532,062 41,533,404 ZFP14 no NA NA1 65,627,570 65,696,043 LEPROT, yes CNV may result in gain-of-function16564564, 17962343, LEPR for LEPROT and gain-of- 18450952, 19854659,function/loss-of-function for LEPR 20624279, 22265003, (severaltranscript variants), 22647716, 23184927, disruption of leptin signalinglinked 23634146, 24239717, to endometriosis, LEPROT is a 24401660,24845415 negative regulator of leptin signaling, LEPR mutations causereduced fertility, LEPROT (and LEPROTL1) involved in growth hormonesignaling, which is implicated in endometrial cancer and endometriosis 3197,001,562 197,065,388 MUC4 yes Complex CNV with 4-step change in17197898, 21349170 copy number and part overlaps with a frequent CNVfound in several endometriosis patients and controls, mucin proteinshave been detected in normal and pathological endometrial tissues (MUC1and MUC4 are major major ones expressed in endometriosis), MUC4 SNPassociated with endometriosis development and endometriosis- relatedinfertility 23 148,575,584 148,608,166 MAGEA11 yes Complex CNV withduplicated and 18048459, 22891251 triplicated regions in a control butCNV is a gain-of-funtion, whereas in the endometriosis patient itloss-of- function (left breakpoint). MAGEA11 is a primate-specific geneand functions as an androgen receptor co-regulator with its mRNA levelsexpressed in a temporal manner in the endometrium, also found toregulate progesterone receptor during human endometrium development 2242,109,998 242,153,935 BOK yes Pro-apoptotic protein and apoptosis is9356461, 19942931, linked to endometriosis, highly 21196342 expressed inovary, testis, and uterus, role in other reproductive biology(preeclampsia, ovarian development) 14 80,613,390 80,649,876 TSHR yesThyroid hormone receptor protein 20691434, 22713859, expression found inmacaques, TSHR 23806847 and thyroid hormone receptors are expressed inhuman endometrium 23 64,731,495 64,811,828 MSN no Implicated inmigration rate in 19095664, 19541800, endometrial stromal cells from22225925, 22272721, endometriosis patients, MSN and 22544491, 23856463,CLDN7 mRNA and protein 24012495 expression found in endometrial cancer,RNAi-mediated knockdown of related family member ezrin (EZR) reducesmigration of endometrial cells in endometriosis, and links toCNV-identified endometriosis candidate TGFB1I1 (via CDKN1A) 2 33,773,80033,903,436 MYADML no Associated with diminished ovarian 11209637,22116950, reserve, which is observed in some 23446861 endometriosispatients 10 104,571,485 104,810,431 CYP17A1 no Gain of whole gene forCYP17A1, 11221867, 15823822, upregulation found in endometrial 18172694,20886547, cancers, conflicting reports of 23609033 genotypes correlatedwwith endometriosis and endometrial cancer 4 159,674,653 159,683,362RXFP1 NA RXFP1 (aka LGR7) mRNA 19416175, 20655530 expression (andrelaxin) are reduced in ectopic endometriotic samples. mRNA expression 730,668,143 30,681,882 CRHR2 no Differential expression found in 23638035eutopic and ectopic endometrium of endometriosis patients. 19 53,252,45753,257,305 PLA2G4C no Involved in endometrial biology and 17459165,21184677, linked to preterm birth; PLA2 22201853, 22658345, enzymes arethe rate-limiting step in 24035605 prostaglandin synthesis andprostaglandins have numerous links endometriosis. 2 24,798,19024,806,680 NCOA1 no NCOA1 (aka SRC1) is linked to PGR 22660634 and isproposed to contribute to pathogenesis of endometriosis along with TNFand MMP9. 7 139,757,225 139,828,667 MKRN1 no Gene family member MKRN3causes 23738509, 23817290 precocious puberty and both endometriosis andprecocious puberty involve GnRH and are treatable with GnRHantagonists/agonists. 9 16,567,785 16,576,265 BNC2 yes Involved inpigmentatation in animals 19956727, 21642636 and in a zebrafish modelresults in female infertility; endometriosis and ovarian cancer areassociated and BNC2 variants have been associated with ovarian cancer. 116,713,074 16,799,710 NBPF1 no NBPF1 interacts with clusterin and20096688, 22211095, 15 PubMed citations for “clusterin 23589125 ANDendometrial” 15 86,943,691 86,944,414 intergenic NA Intergenic, 21Kbupstream of AEN, 18264133, 21196342 an apoptosis gene and apoptosis islinked to endometriosis 15 86,941,339 86,943,690 intergenic NAIntergenic, 21Kb upstream of AEN, 18264133, 21196342 an apoptosis geneand apoptosis is linked to endometriosis21Kb upstream of AEN, anapoptosis gene and apoptosis is linked to endometriosis 2 191,869,063191,873,037 MYO1B no Actin expression levels linked to 15475577,20080738, endometriosis and actin; MYO1B and 20471271, 22878529, actinlinks 22735530 2 191,873,038 191,874,236 MYO1B no Actin expressionlevels linked to 15475577, 20080738, endometriosis and actin; MYO1B and20471271, 22878529, actin links 22735530 6 47,731,384 47,734,315 GPR111no NA 22837050 6 70,290,311 70,295,413 intergenic NA NA NA

Example 2

Pathway analysis software may be used to identify whether the candidategene is a drug target, which may be FDA-approved, in clinical trials oramenable for development of a new therapeutic. Such information willassist in the design of clinical trials (e.g., patient stratificationfor genetic subtypes) or will be used to facilitate clinical trials thatare in progress, thereby reducing the attrition rate (failure to receiveFDA approval) and reducing the time and cost of drug development. When acandidate EN gene is identified as a known drug target of anFDA-approved therapeutic, the drug can be repurposed and approved foruse in a new indication (e.g., a cancer or anti-inflammatory agent maybe beneficial to EN patients as well). Those skilled in the art willrecognize that Phase II and III failures may be rescued with additionalclinical trial data that accounts for genetic subtypes, particularlywhen the drug fails for lack of efficacy. For example, if a drug will bedesigned or established to target a particular gene defect (e.g., use ofan RNAi therapeutic to decrease aberrant overexpression of the gene thatis caused by a CNV or other type of genetic variant), it will beexpected that only EN patients with that particular genetic subtype willbenefit from the targeted therapy.

Example 3

Once a region is identified that has one or more genetic variationsassociated with EN, probes and/or primers can be prepared to furthercharacterize those variations in test subjects. Targeted sequencing ofCNV-identified regions/genes that are associated with EN (FIG. 2 )and/or found to have EN-relevant biology (Table I) is a preferredembodiment for further characterization of the genetic findings.Targeted sequencing enables ascertainment of the mutational spectrum inEN patients that are associated with EN. Targeted sequencing can beperformed using one or more methods known to those skilled in the artsuch as, but not limited to, Sanger sequencing of PCR amplified regions,high-throughput sequencing of specific regions of interest, exomesequencing, or whole genome sequencing. Known and novel variants(SNPs/SNVs/indels) identified in sequencing experiments and/or data canbe interpreted (mainly for exonic variants, but some adjacent intronicsequence variant data is also available) using NCBI's dbSNP, the ExomeVariant Server (EVS) hosted by a website at the University of Washington(evs.gs.washington.edu/EVS/), or the Exome Aggregation Consortium (ExAC)browser hosted by the Broad Institute (http://exac.broadinstitute.org/)to assess their frequency in the general population. In anotherembodiment, interpretation of EN-associated regions/genes identified onthe basis of CNVs can be performed on prexisting exome or whole genomesequence data (i.e., targeted interpretation). It can be appreciated bythose skilled in the art that the genome search space for identifyingother EN-associated variants is dramatically reduced by performingtargeted sequencing and/or targeted interpretation of CNV-identifiedregions of interest (e.g., such analyses may involvesequencing/interpretation of variants present in only 20-30 EN candidategenes as opposed to variants present in all approximately 20,000 genesin the human genome). As described herein, genetic variations in TGFBR3and PDE1C were found to be associated with EN (FIG. 2 ). TGFBR3 is oneof several TGF beta superfamily members involved in endometrial andplacental function, which includes activin/inhibin regulation. TGFBR3 isa proteoglycan that functions as a co-receptor for inhibins (INHA,INHBs) and TGF beta isoforms (TGFBs). TGFBR3 and INHA are down-regulatedin endometrial carcinoma, and TGFBR3 is down-regulated in 90% breastcancers (via LOH or at the protein level). Restoring TGFBR3 expressioninhibits tumor invasiveness, angiogenesis, and metastasis. Along withARRB2, TGFBR3 regulates integrin α5β1 (ITGA5/ITGB1) trafficking andfocal adhesion formation, which are processes implicated inendometriosis.

PDE1C has high affinity for cAMP and cGMP and is a calmodulin-dependentphosphdiesterase. PDE1C regulates collagen homeostasis and is a keyregulator of pathological vascular remodeling. PDE1C (HG18;chr7:31759157-32304908) maps to a linkage peak found in familialendometriosis.

Example 4

The CNV analysis described herein (Example 1) revealed the presence of a4-probe spanning heterozygous TGFBR3 deletion in 3 individuals withendometriosis (FIG. 5 ). The deletion is within an intron of TGFBR3 andwas predicted, on the basis of the array data, to be at least 6.8 kb insize. Since the deletion was observed in 3 unrelated females, it wassuspected that either the deletion occurs recurrently in differentfamilies; or the deletion represents an individual mutation thatoccurred some time ago and is present in descendants of the individualin whom it originally occurred (founder effect).

To determine whether the deletion was associated with EN or was anartifact and to map precise breakpoints at each end of the deletion, inorder to define the size, the following experiments were conducted: 3pairs of PCR primers were designed to amplify putative products of agiven size only in those individuals who carried the deletion. One ofthese primer pairs successfully generated a product of the expected sizein the deletion carriers but not in either normal DNA or DNA from anendometriosis patient without the deletion. The primer pair that hadsuccessfully generated a product in the deletion carriers was used againto amplify a product in all three carriers, for the purpose of Sangersequencing. Data from Sanger sequencing revealed confirmation of thepresence of the deletion in all 3 carriers; the precise sequence at eachbreakpoint; the precise size of the deletion (8,071 bp); and thepresence of an extra 2 bp between the endpoints (‘GG’).

This data may be used to generate an even more efficient assay of thepresence of the deletion in endometriosis cohorts and will be used asboth a screening tool and a diagnostic test.

Methods and Results

3 sets of primers were tested:

OUTER_FWD 57.21 TTTTTGGTTAAACCCTACATCAC (SEQ ID NO: 150) OUTER_REV 59.52TCCCTAGCCCATTTCTAAATCTT (SEQ ID NO: 151) MIDDLE_FWD 59.55TCCCTTGCCAGTGGAACTAT (SEQ ID NO: 152) MIDDLE_REV 59.99TCGGCCAGAAGAGTCTGTTT (SEQ ID NO: 153) INNER_FWD 60.05TGAATTTCTGGGCATGTGAA (SEQ ID NO: 154) INNER_REV 60.13GAGAGGCCTGAGCACAAAAG (SEQ ID NO: 155)

Only primers OUTER_FWD and OUTER_REV generated a product that wasspecific to the deletion carriers (and yielded negative results innormal DNA and in an individual with endometriosis who did not carry adeletion, based on the 1M array data). These PCR primers were used toamplify a product from all 3 deletion carriers, for the purpose ofSanger sequencing (FIG. 7 ).

The three products shown in FIG. 7 were purified and subjected to Sangersequencing using the Fwd and Rev primers as sequencing primers. Sequencefrom the 3 products was queried, using BLAT, at the UCSC server(http://genome.ucsc.edu/cgi-bin/hgBlat?command=start). In each case, thesequence obtained with the Fwd sequencing primers clearly demonstratedthe presence of an approximately 8 kb deletion, with apparentlyidentical breakpoints in all 3 individuals (FIG. 8 ). Further analysisof the sequences revealed the deletion size to be 8,071 bp (whichencompassed the expected 4 probes from the Agilent 1M feature (design ID021529)) and was identical in all 3, unrelated, individuals.

The sequences (hg19 coordinates) obtained were as follows:

>1739.Forward (SEQ ID NO: 156)TAATCGACTCATGCGGTGATTGGGAATTCTTTCAGGGCAACAGGCAATGTGTTAAATATGCACTGTTGAGTACACTGTGCAAAGTTATGAAATTCTCTCTTTCCCTCCTGACATTTTTTTTTCCAAGTACTTCACTGGCTACTCCAGAAGCAAAGGAATAGAGAAAAGAGTGAAATCAGAACTAGTGAGTGGACTTGGTTACTGTAAGATCACTGGTAAAAGTCTGAAAGAAACAAAGGTGGAGCAAATTCAAAATGGATCAGATGTGTGTACACATGTATCAACAAATAGAAGTTAAGCCATAATGGGCACAAGGGGACACTTCAGCTCCGGGCAAGAGTTAGGCTATGGTAGTGACCTTGGATCCTAAAGCTGGGCTCTGTCCTTGCTTCACAGTGAGAATCAGTAACACCTCATCTCATTAGCTCTCTTATCTTCAAAAGTATCCAAGTCATACCTGTAATTTGCCCCTCATCCTCCAAGAGTTGTACAAATTTCAGGTTCAGCTGAAGGACTCTGTGGTTCAGGTGAAAAAAAAAGCCATAAATACAAAGCATTATTGTAGGGTGCTTTGGACTAGAACCCTGTCTAATATCTGGGCCTTGATATTTCAGCCTTTCAGACAAGGCCAAGGAGCTCAGAGACAAGGACTCCTTCAATCAGCCAGCAGTGCCACTGAGGTGCCCCGGCGGGCTGGACAGGAAAGCATGGAGAACATGGCTGCAATGGAAGCCAAAGCAGCAGGTCTTCCAAACACAGACTCAGATGCCTGTGTCTTTAAGACCAGACCCTCATAAATGGATTGCTTCTGCTGGACACCACGCTCTAAATAAACAGACTCTTCTGGCCGACACACAACTTCCTGTAGGATTCTGGGGGGGTAAAGCTTGAAAAGGCTGCCAAATCCAATGACCAGCAACTTTTGAGCTGACTTAGAAAACAAGCTACAAAGACTTGAGTCCAGAGTAAACAAAGGAAAAAGCCATATTAAACAGGGAACAAATTACTATATCGGCAGGGAAATTTTAA >2732.Forward (SEQ ID NO: 157)TAATCGACTCATGCGGTGATTGGGAATTCTTTCAGGGCAACAGGCAATGTGTTAAATATGCACTGTTGAGTACACTGTGCAAAGTTATGAAATTCTCTCTTTCCCTCCTGACATTTTTTTTTCCAAGTACTTCACTGGCTACTCCAGAAGCAAAGGAATAGAGAAAAGAGTGAAATCAGAACTAGTGAGTGGACTTGGTTACTGTAAGATCACTGGTAAAAGTCTGAAAGAAACAAAGGTGGAGCAAATTCAAAATGGATCAGATGTGTGTACACATGTATCAACAAATAGAAGTTAAGCCATAATGGGCACAAGGGGACACTTCAGCTCCGGGCAAGAGTTAGGCTATGGTAGTGACCTTGGATCCTAAAGCTGGGCTCTGTCCTTGCTTCACAGTGAGAATCAGTAACACCTCATCTCATTAGCTCTCTTATCTTCAAAAGTATCCAAGTCATACCTGTAATTTGCCCCTCATCCTCCAAGAGTTGTACAAATTTCAGGTTCAGCTGAAGGACTCTGTGGTTCAGGTGAAAAAAAAAGCCATAAATACAAAGCATTATTGTAGGGTGCTTTGGACTAGAACCCTGTCTAATATCTGGGCCTTGATATTTCAGCCTTTCAGACAAGGCCAAGGAGCTCAGAGACAAGGACTCCTTCAATCAGCCAGCAGTGCCACTGAGGTGCCCCGGCGGGCTGGACAGGAAAGCATGGAGAACATGGCTGCAATGGAAGCCAAAGCAGCAGGTCTTCCAAACACAGACTCAGATGCCTGTGTCTTTAAGACCAGACCCTCATAAATGGATTGCTTCTGCTGGACACCACGCTCTAAATAAACAGACTCTTCTGGCCGACACACAACTTCCTGTAGGATTCTGGGTGGGTAAAGCTTGAAAAGGCTGCCAAATCCAATGACCAGCAACTTTTGAGCTGACTTAGAAAACAAGCTACAAAGACTTGAGTCCAGAGTAAACAAAGGAAAAAGCCATATTAAACAGGGAACAAATTACTATATCGGCAGGGATATTTTAA >3697.Forward (SEQ ID NO: 158)TAATCGACTCATGCGGTGATTGGGAATTCTTTCAGGGCAACAGGCAATGTGTTAAATATGCACTGTTGAGTACACTGTGCAAAGTTATGAAATTCTCTCTTTCCCTCCTGACATTTTTTTTTCCAAGTACTTCACTGGCTACTCCAGAAGCAAAGGAATAGAGAAAAGAGTGAAATCAGAACTAGTGAGTGGACTTGGTTACTGTAAGATCACTGGTAAAAGTCTGAAAGAAACAAAGGTGGAGCAAATTCAAAATGGATCAGATGTGTGTACACATGTATCAACAAATAGAAGTTAAGCCATAATGGGCACAAGGGGACACTTCAGCTCCGGGCAAGAGTTAGGCTATGGTAGTGACCTTGGATCCTAAAGCTGGGCTCTGTCCTTGCTTCACAGTGAGAATCAGTAACACCTCATCTCATTAGCTCTCTTATCTTCAAAAGTATCCAAGTCATACCTGTAATTTGCCCCTCATCCTCCAAGAGTTGTACAAATTTCAGGTTCAGCTGAAGGACTCTGTGGTTCAGGTGAAAAAAAAAGCCATAAATACAAAGCATTATTGTAGGGTGCTTTGGACTAGAACCCTGTCTAATATCTGGGCCTTGATATTTCAGCCTTTCAGACAAGGCCAAGGAGCTCAGAGACAAGGACTCCTTCAATCAGCCAGCAGTGCCACTGAGGTGCCCCGGCGGGCTGGACAGGAAAGCATGGAGAACATGGCTGCAATGGAAGCCAAAGCAGCAGGTCTTCCAAACACAGACTCAGATGCCTGTGTCTTTAAGACCAGACCCTCATAAATGGATTGCTTCTGCTGGACACCACGCTCTAAATAAACAGACTCTTCTGGCCGACACACAACTTCCTGTAGGATTCTGGGGGGGTAAAGCTTGAAAAGGCTGCCAAATCCAATGACCAGCAACTTTTGAGCTGACTTAGAAAACAAGCTACAAAGACTTGAGTCCAGAGTAAACAAAGGAAAAAGCCATATTAAACAGGGAAC AAATTAC

1739, 2732 and 3697 refer to anonymized identifiers for the 3endometriosis cases.

These sequences, once analyzed using the UCSC server (using BLAT), werefound to break down into the following subsequences:

>1739.Forward (SEQ ID NO: 159)TAATCGACTCATGCGGTGATTGGGAATTCTTTCAGGGCAACAGGCAATGTGTTAAATATGCACTGTTGAGTACACTGTGCAAAGTTATGAAATTCTCTCTTTCCCTCCTGACATTTTTTTTTCCAAGTACTTCACTGGCTACTCCAGAAGCAAAGGAATAGAGAAAAGAGTGAAATCAGAACTAGTGAGTGGACTTGGTTACTGTAAGATCACTGGTAAAAGTCTGAAAGAAACAAAGGTGGAGCAAATTCAAAATGGATCAGATGTGTGTACACATGTATCAACAAATAGAAGTTAAGCCATAATGGGCACAAGGGGACACTTCAGCTCCGGGCAAGAGTTAGGCTATGGTAGTGACCTTGGATCCTAAAGCTGGGCTCTGTCCTTGCTTCACAGTGAGAATCAGTAACACCTCATCTCATTAGCTCTCTTATCTTCAAAAGTATCCAAGTCATACCTGTAATTTGCCCCTCATCCTCCAAGAGTTGTACAAATTTCAGGTTCAGCTGAAGGACTCTGTGGTTCAGGTGAAAAAAAAAGCCATAAATACAAAGCATTATTGTAGGGTGCTTTGGACTAGAACCCTGTCTAATATCTGGGCCTTGATATTTCAGCCTTTCAGACAAGGCCAAGGAGCTCAGAGACAAGGACTCCTTCAATCAGCCAGCAGTGCCACTGAGGTGCCCCGGCGGGCTGGACAGGAAAGCATGGAGAACATGGCTGCAATGGAAGCCAAAGCAGCAGGTCTTCCAAACACAGACTCAGATGCCTGTGTCTTTAAGACCAGACCCTCATAAATGGATTGCTTCTGCTGGACACCACGCTCTAAATAAACAGACTCTTCTGGCCGACACACAACTTCCTGTAGGATTCTGGGGGGGTAAAGCTTGAAAAGGCTGCCAAATCCAATGACCAGCAACTTTTGAGCTGACTTAGAAAACAAGCTACAAAGACTTGAGTCCAGAGTAAACAAAGGAAAAAGCCATATTAAACAGGGAACAAATTACTATATCGGCAGGGAAATTTTAA >2732.Forward (SEQ ID NO: 160)TAATCGACTCATGCGGTGATTGGGAATTCTTTCAGGGCAACAGGCAATGTGTTAAATATGCACTGTTGAGTACACTGTGCAAAGTTATGAAATTCTCTCTTTCCCTCCTGACATTTTTTTTTCCAAGTACTTCACTGGCTACTCCAGAAGCAAAGGAATAGAGAAAAGAGTGAAATCAGAACTAGTGAGTGGACTTGGTTACTGTAAGATCACTGGTAAAAGTCTGAAAGAAACAAAGGTGGAGCAAATTCAAAATGGATCAGATGTGTGTACACATGTATCAACAAATAGAAGTTAAGCCATAATGGGCACAAGGGGACACTTCAGCTCCGGGCAAGAGTTAGGCTATGGTAGTGACCTTGGATCCTAAAGCTGGGCTCTGTCCTTGCTTCACAGTGAGAATCAGTAACACCTCATCTCATTAGCTCTCTTATCTTCAAAAGTATCCAAGTCATACCTGTAATTTGCCCCTCATCCTCCAAGAGTTGTACAAATTTCAGGTTCAGCTGAAGGACTCTGTGGTTCAGGTGAAAAAAAAAGCCATAAATACAAAGCATTATTGTAGGGTGCTTTGGACTAGAACCCTGTCTAATATCTGGGCCTTGATATTTCAGCCTTTCAGACAAGGCCAAGGAGCTCAGAGACAAGGACTCCTTCAATCAGCCAGCAGTGCCACTGAGGTGCCCCGGCGGGCTGGACAGGAAAGCATGGAGAACATGGCTGCAATGGAAGCCAAAGCAGCAGGTCTTCCAAACACAGACTCAGATGCCTGTGTCTTTAAGACCAGACCCTCATAAATGGATTGCTTCTGCTGGACACCACGCTCTAAATAAACAGACTCTTCTGGCCGACACACAACTTCCTGTAGGATTCTGGGTGGGTAAAGCTTGAAAAGGCTGCCAAATCCAATGACCAGCAACTTTTGAGCTGACTTAGAAAACAAGCTACAAAGACTTGAGTCCAGAGTAAACAAAGGAAAAAGCCATATTAAACAGGGAACAAATTACTATATCGGCAGGGATATTTTAA >3697.Forward (SEQ ID NO: 161)TAATCGACTCATGCGGTGATTGGGAATTCTTTCAGGGCAACAGGCAATGTGTTAAATATGCACTGTTGAGTACACTGTGCAAAGTTATGAAATTCTCTCTTTCCCTCCTGACATTTTTTTTTCCAAGTACTTCACTGGCTACTCCAGAAGCAAAGGAATAGAGAAAAGAGTGAAATCAGAACTAGTGAGTGGACTTGGTTACTGTAAGATCACTGGTAAAAGTCTGAAAGAAACAAAGGTGGAGCAAATTCAAAATGGATCAGATGTGTGTACACATGTATCAACAAATAGAAGTTAAGCCATAATGGGCACAAGGGGACACTTCAGCTCCGGGCAAGAGTTAGGCTATGGTAGTGACCTTGGATCCTAAAGCTGGGCTCTGTCCTTGCTTCACAGTGAGAATCAGTAACACCTCATCTCATTAGCTCTCTTATCTTCAAAAGTATCCAAGTCATACCTGTAATTTGCCCCTCATCCTCCAAGAGTTGTACAAATTTCAGGTTCAGCTGAAGGACTCTGTGGTTCAGGTGAAAAAAAAAGCCATAAATACAAAGCATTATTGTAGGGTGCTTTGGACTAGAACCCTGTCTAATATCTGGGCCTTGATATTTCAGCCTTTCAGACAAGGCCAAGGAGCTCAGAGACAAGGACTCCTTCAATCAGCCAGCAGTGCCACTGAGGTGCCCCGGCGGGCTGGACAGGAAAGCATGGAGAACATGGCTGCAATGGAAGCCAAAGCAGCAGGTCTTCCAAACACAGACTCAGATGCCTGTGTCTTTAAGACCAGACCCTCATAAATGGATTGCTTCTGCTGGACACCACGCTCTAAATAAACAGACTCTTCTGGCCGACACACAACTTCCTGTAGGATTCTGGGGGGGTAAAGCTTGAAAAGGCTGCCAAATCCAATGACCAGCAACTTTTGAGCTGACTTAGAAAACAAGCTACAAAGACTTGAGTCCAGAGTAAACAAAGGAAAAAGCCATATTAAACAGGGAAC AAATTAC

In each case:

The first part of the sequence maps to: chr1:92240239-92240474 (hg19)The second part of the sequence maps to: chr1:92248545-92249312 (hg19)The precise deletion size is calculated as start of second segment—endof first segment: 92248545−92240474=8,071 bp

The presence of the dinucleotide ‘GG’ in between these two sequences islikely a product of the type of event/mechanism that resulted in thedeletion.

The outer primers defined above would be expected to generate a productof 9,499 bp (chr1:92,240,188-92,249,686) from a normal, wild-typegenomic sequence, which does not harbor the deletion. In the 3 casesdescribed above, the deletion is present heterozygously, so a wild-type,non-deleted allele is also present. A 9,499 bp band was not observed inFIG. 7 because amplification of PCR products in this size range requiresspecific, special conditions (long extension times, use of specialpolymerases etc). In standard PCR, products of 9,499 bp will not amplifyefficiently and will remain unobserved on DNA gels. The reason that PCRacross a deletion can be used as a screening tool is that the primerscan be designed to generate a small PCR product only when a deletion ispresent, a product that cannot be generated from wild-type sequences, inwhich the primers are too far apart. Note that, in FIG. 7 , theapproximate size of the PCR product is 1.5 kb, which is consistent withthe sequencing results:

-   -   Wild-type PCR product would be 9,499 bp (about 9.5 kb)    -   Outer primer PCR product about 1.5 kb    -   Deletion size=8,071 bp, and    -   about 8 kb deletion+1.5 kb PCR product size=about 9.5 kb        wild-type primer distance.

Hence, the outer primer sequences described represent a screening toolthat can be used diagnostically in endometriosis. By screeningindividuals with endometriosis using the 2 outer primers and standardPCR conditions, the presence of an approximately 1.5 kb band confirmsthe presence of the deletion. The deletion includes a transcriptionfactor binding site.

However, other primers may be employed, e.g., as a pair or in a nestedset of primers, to allow for a smaller product to be generated (for morerapid PCR). In one embodiment, the primer pair is capable of generatinga diagnostic product even when pooled samples are tested (e.g., thepresence of 1/10 individuals with a deletion will result in generationof a product, therefore reporting the presence of at least one deletionwithin the 10 individuals being tested). This will be a useful screeningtest, which will allow thousands of individuals to be screened veryrapidly (for example, 1,000 individuals can be screened using 100 PCRreactions, each containing DNA from 10 individuals). Any pool that ispositive can be ‘split’ and the individual cases tested singly, in orderto identify the individual(s) from whom the band originates. Pools thatare negative would not need to be ‘split’ in this way for furtheranalysis.

For example, using standard PCR conditions, PCR may be performed using apair of primers designed to generate a product if and only if at leastone of the individual's TGFBR3 genes possesses a deletion, for instance,using the primers that yielded the product in FIG. 7 . If the band ispresent, the patient has the deletion, indicating EN or a predispositionor susceptibility thereto. If the band is absent, the patient does nothave the deletion and so does not have this marker for EN. In a furtherrefinement of the method to detect the deletion, a new set of primerswas designed. The new set of primers, Endo_screen_F=GGAATTCTTTCAGGGCAACA(SEQ ID NO: 162) and Endo_screen_R=GACAGAGCCCAGCTTTAGGA (SEQ ID NO:163), generate a product of size 362 bp when the deletion is present.Additionally, a ‘control’ set of primers was designed to amplify thesequence within the deletion, in order to differentiate del/delhomozygotes from del/wt heterozygotes. This ‘controls’ primer pair wasTGFBR3_internal_F=TTGTGTGGCCTCACTCAAAC (SEQ ID NO: 164) andTGFBR3_internal_R=CCCTTGACTTGCTGTGAGGT (SEQ ID NO: 165), and generates a460 bp product when at least one copy of the sequence internal to thedeletion is present. The 460 bp ‘control’ band can be easilydifferentiated from the 362 bp product created when the deletion ispresent. Thus, using a combination of both primers in the PCR reaction,there are 3 possible outcomes: 460 bp band only→wt/wt (no deletion); 460bp+362 bp bands→wt/del (heterozygous deletion is present); 362 bp bandonly→del/del (homozygous deletion). This primer combination was used toscreen a total of 911 endometriosis samples (100 discovery samples plus811 unrelated individuals). A total of 5 heterozygous deletion carrierswere found, 3 from the original discovery cohort of 100 (thisrepresented a technical validation) plus 2 further, new, cases. Thus,the heterozygous deletion was present in a total of 5/911 cases=0.55%.In a total of 2,257 non-endometriosis cases, only one positive was found(in a male for whom no family history is available). Thus, the Fisher'sexact test for this deletion is FET=0.0089 and the odds ratio isOR=12.45. FIG. 11 represents an example of a typical result in aheterozygous deletion carrier and a non-carrier (these were the only 2genotypes seen—no homozygous carriers have been observed but aretheoretically predicted to exist, albeit rarely).

Example 5

For each CNV listed in FIG. 1 , the relevant intron(s)/exon(s) sequencefor the CNV was obtained from the consensus HG18 sequence. The sequencesin the files are for complete introns/exons, rather than the specificcomponent relevant to the CNV. CNVs that encompass consecutive intronsand exons contain multiple features reported per CNV.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

1-19. (canceled)
 20. A method comprising: (a) (i) hybridizing a nucleicacid probe to a polynucleic acid, wherein the polynucleic acid is from asample from a human female subject with endometriosis, or (ii)synthesizing a nucleic acid product from a polynucleic acid, wherein thepolynucleic acid is from a sample from a human female subject withendometriosis; and (b) detecting in the polynucleic acid at least onegenetic variation comprising a copy number variation (CNV) from a panelof low frequency genetic variations; wherein detecting comprisesdetecting by PCR, sequencing, nucleic acid hybridization, microarrayanalysis or northern blot or a combination thereof, wherein the panel oflow frequency genetic variations comprises at least one low frequencygenetic variation for each of a plurality of genetic loci, and whereinthe at least one low frequency genetic variation occurs at a frequencyof 0.1% or less in a population of female subjects without a diagnosisof endometriosis.
 21. The method of claim 20, wherein the CNV introducesa breakpoint in a gene.
 22. The method of claim 20, wherein detectingcomprises detecting at least two genetic variations.
 23. The method ofclaim 20, wherein the CNV occurs in an exon of a gene.
 24. The method ofclaim 23, wherein the gene is selected from the group consisting of:HMGB3, IMPK, ACCS, PTK2, MUC4, MAGEA11, MSN, MYADML, CRHR2, PLA2G4C,NCOA1, and BNC2.
 25. The method of claim 24, wherein the CNV comprises asequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:31, SEQ IDNO: 33 and SEQ ID NO:
 35. 26. The method of claim 23, wherein the CNVdisrupts a function of at least two genes.
 27. The method of claim 26,wherein the CNV comprises a sequence selected from the group consistingof: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ IDNO: 29, SEQ ID NO: 32, SEQ ID NO:
 34. 28. The method of claim 20,wherein the CNV occurs in an intron of a gene or an intergenic region.29. The method of claim 28, wherein the CNV comprises a sequenceselected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 21and SEQ ID NO:
 30. 30. The method of claim 20, wherein the polynucleicacid comprises blood, saliva, urine, serum, tears, skin, tissue or hairfrom the human female subject.
 31. The method of claim 20, whereindetecting comprises detecting by multiplex ligation-dependent probeamplification (MLPA), molecular beacon, array Comparative GenomicHybridization, Invader assay, ligase chain reaction (LCR), fluorescencein situ hybridization, or any combination thereof.
 32. The method ofclaim 20, wherein detecting comprises detecting by whole genomesequencing or whole exome sequencing.
 33. The method of claim 20,wherein the method further comprises administering a pharmaceuticalcomposition for treating endometriosis to the subject.
 34. The method ofclaim 20, wherein the human female subject is asymptomatic or isinfertile.
 35. The method of claim 24, wherein the CNV causes loss offunction of a PLA2G4C gene.
 36. The method of claim 23, wherein the geneis selected from group consisting of LEPR, CNNM2, MKRN1 and SLC37A3. 37.The method of claim 32, wherein the detecting further comprises anin-silico analysis.
 38. The method of claim 20, wherein the detectingcomprises detecting a first genetic variation, wherein the first geneticvariation is the CNV of the PLA2G4C gene, wherein the first geneticvariation and a second genetic variation are in a panel comprising twoor more genetic variations.